Fig 2: (A) Representative phase contrast image of HIO-derived epithelial cells seeded into the Chip. Cells were exposed to continuous media flow at 30 µL/h and imaged after 0, 3, 5, and 7 days. Static Chip was imaged after 7 days. Scale bar = 200 µm. (B) Stitched phase contrast image of HIO-epithelial cells that were exposed to continual media flow at 30 µL/h for 5 days. Scale bar = 1 mm. (C) Representative brightfield image of cross-section of Chip that was exposed to continual media flow at 30 µL/h for 14 days. Scale bar = 250 µm. Representative fluorescent images showing (D) E-cadherin/CDH1 (red), ZO-1 (green), DAPI (blue), and (E) E-cadherin (blue), CDX2 (red), and Villin (green), in cross-section of Chip under conditions similar to C. Both scale bars = 50 µm. (F) Representative fluorescent images showing E-cadherin/CDH1 (red), and MUC2, lysozyme, FABP2, and chromogranin A (all green), counterstained with DAPI (blue), in cross-section of Chips that were exposed to continual media flow at 30 µL/h for 7 days. Scale bar = 10 µm. (G) Representative images of in situ hybridization for LGR5+ and WDR43+ (white arrows) in conditions similar to F. Scale bar = 10 µm. (H) Representative fluorescent image showing E-cadherin/CDH1 (blue), Ki67 (green), and DAPI (blue) in conditions similar to F. Scale bar = 100 µm. Graph shows percent Ki67+ nuclei per section.

Fig 3: Gln activated G6PD activity and stabilized AGR2 redox homeostasis via the HBP(A) Hexosamine biosynthetic pathway. (B, C) Effects of Gln and glucosamine on G6PD protein and O-GlcNAcylation levels as determined by immunoblotting of G6PD protein with antibodies against the G6PD protein and O-GlcNAc. HT-29 CL.16E cells were treated with 2 mM Gln or 10 mM glucosamine for 12 h, and cultured in the absence or presence of DON (20 µM). (D) Immunoblot detection of cellular protein O-GlcNAcylation. HT-29 CL.16E cells were exposed for 12 h to LPS (100 ng/ml), cultured in the absence or presence of Gln (2 mM), and then stimulated with or without Don (N = 3 per group). (E) Chemical enzyme labeling and biotinylation to detect the O-GlcNAcylation of G6PD (N = 3 per group). (F) Immunoblot detection of G6PD dimers and monomers. Cells were cross-linked with 1 mM DSS (N = 3 per group). (G) A -SulfoBiotics- Protein Redox State Monitoring Kit was used to determine the thiol redox state of recombinant proteins (N = 3 per group). (H) S-glutathionylated AGR2/AGR2 was measured by immunoblot analysis (N = 3 per group).(I) Immunoblot detection of mature MUC2 and immature MUC2 protein expression (N = 3 per group). * Compared with control, & Compared with the LPS group, & P < 0.05; &&P < 0.01; &&& P < 0.001. # Compared with LPS + Gln group, #P < 0.05; # #P < 0.01; # # #P < 0.001.

Fig 4: Patients 1 and 2 show severe loss of goblet cells in the small intestines. (A) Patient small bowel was nearly devoid of goblet cells on H&E staining of formalin-fixed paraffin-embedded (FFPE) sections, with few potential examples of apical staining clearances consistent with the morphology and localization of goblet cell thecae (A-1). The section also shows disorganization of columnar epithelial cells (A-2). Mitotic figures (A-3) and regenerative crypts (arrows) indicate a regenerative state. Apoptotic bodies also are present (A-4), indicating ongoing apoptotic activity. Paneth cells appeared unremarkable (black arrowheads). White arrowheads point to the features highlighted in each respective inset. Images were acquired using a slide scanner with a 40× objective and were cropped digitally. Scale bars: 100 µm (main panel); 10 µm (magnified insets). Digital zoom was used for magnified insets. Representative section from patient 1 is shown here. Both patients showed identical presentation. (B) Quantification of goblet cell depletion in small-bowel H&E sections. Results are the ratio of goblet cells to total epithelial cells. Each point represents the data from 1 individual. Line represents the median and bars indicate 95% CI. (C) Fluorescent immunohistochemistry staining for the goblet cell markers TFF3 (green, clone B-1), CLCA1 (red, clone EPR12254-88), and nonglycosylated MUC2 precursor (turquoise, clone CCP58) in paraffin-embedded sections from small bowel detected by a laser scanning microscope using a 10× objective. Nuclei were stained with Hoechst (blue). Signal was acquired from the entire thickness of the section by adjusting the pinhole to cover 9 µm. Each marker was replicated at least 3 independent times. CLCA1 was visualized using a goat anti-rabbit AF647 antibody and goat anti-mouse AF546 for TFF3. Nonglycosylated MUC2 antibody was conjugated directly with AF594. For small bowel, TFF3 staining was replicated across 4 noninflamed controls and 5 inflamed controls, while CLCA1 and nonglycosylated MUC2 precursor staining were replicated across 2 noninflamed controls and 2 inflamed controls. Small-bowel inflamed control shown in the figure is from a patient diagnosed with Familial Mediterranean fever.

Fig 5: S-glutathionylation of AGR2 restricted the processing of MUC2.(A) AGR2 was immunoprecipitated from HT-29 CL.16E cells, and associated MUC2 was detected by immunoblotting. Rabbit or mouse IgG was used as a control. A total of 5% of the volume of lysate used for the immunoprecipitation was run on the gel for comparison (input). (B) Immunofluorescent localization of the MUC2 precursor (green) and AGR2 (red) in HT-29 CL.16E cells. Blue indicates nuclear (DAPI) staining. Scale bar: 10 µm. (C) The expression levels of the MUC2 precursor, mature MUC2 and AGR2 were assessed in AGR2 Knockout HT-29 CL.16E cell line. (D) Immunofluorescence staining for the MUC2 precursor (green) and mature MUC2(red) in AGR2 Knockout HT-29 CL.16E cell line. Blue indicates nuclear (DAPI) staining. Scale bar: 10 µm. (E) Expression of mature MUC2, immature MUC2, AGR2, GRP78, and CHOP in the distal colons of sham, burn–infected mice at 5 days post-injury (N = 5 per group). (F) Staining for ER-localized MUC2 precursor (green) in mouse goblet cells to assess MUC2 biosynthesis at 5 days post-injury. Scale bar: 50 µm. (G) Representative confocal microscopy images of distal colon sections for analysis of AGR2 (red) and MUC2 (green) at 5 days post-injury, with yellow arrows representing immature MUC2 and red arrows indicating mature MUC2. Scale bar: 50 µm. (H) Cysteine thiol levels of AGR2 in the distal colons of sham and burn-infected mice at 5 days post-injury. Tissue lysates were prepared, incubated with Protein-SHifter Plus, and subjected to SDS?PAGE without BME (N = 3 per group). (I) Immunoblotting for S-glutathionylation of protein in colon tissues (DTT, negative control; GAPDH, loading control; N = 4 per group). (J) Co-IP showing S-glutathionylated AGR2 in colon tissue lysates from sham or burn-infected mice at 5 days post-injury (IP, GSH; IB, AGR2). (K) Tissue lysates were incubated with a MUC2-VTNR antibody coupled to Thermo Scientific Pierce Protein A/G magnetic beads for 24 h. The precipitates were subjected to Western blotting with anti-MUC2-VTNR and anti-AGR2 antibodies. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)