A sentinel goblet cell guards the colonic crypt by triggering Nlrp6-dependent Muc2 secretion by George M. H. Birchenough, Elisabeth E. L. Nyström, Malin E. V. Johansson, and Gunnar C. Hansson Science Volume 352(6293):1535-1542 June 24, 2016 Published by AAAS

Fig. 1 Select TLR ligands induce Muc2 secretion in distal colon. Select TLR ligands induce Muc2 secretion in distal colon. In (A) to (G), intestinal explants were treated with TLR ligands or CCh. (A and B) Quantification of mucus growth from distal colonic (A) or ileal (B) explants. Unt., untreated; Flag., treated with flagellin. (C) Potential difference (PD) measurement: solid lines, mean; dashed lines, SEM. (D) Confocal micrographs of RedMUC298trTg tissue. Gray, actin; red, mCherry-MUC2. (E) Confocal z-stacks of tissue and 1-μm beads. Blue, tissue; red, beads; im, impenetrable mucus. (F) Impenetrable mucus thickness. (G) Concentration-response curves. Red dashed line, lipid A EC50; black dashed lines, estimated mucus and stool lipid A concentrations. (H) LAL reactivity of stool, mucus, and lipid A EC50 (0.85 μM). (I) Quantification of 16S rRNA genes in stool and mucus by quantitative polymerase chain reaction. Data are means ± SEM (n = 4 or 5); *P < 0.05, Dunnett test [(A), (B), (H)], Sidak test (F), or Mann-Whitney test (I). Scale bars, 50 μm. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS

Fig. 2 TLR ligand–responsive GCs are localized to the upper crypt. TLR ligand–responsive GCs are localized to the upper crypt. Colonic explants were treated with TLR ligands or CCh. (A) Confocal micrographs of cryosections from RedMUC298trTg tissue. Red, MUC2; blue, DNA. (B) Upper crypt (yellow boxes) or lower crypt (green boxes) magnified from (A); a dashed gray line shows the epithelial surface. (C and D) Quantification of mucus growth rates. (E) Upper-crypt GCs (yellow arrowheads) and intercrypt GCs (green arrowheads) in RedMUC298trTg colon. Upper left: Confocal z-stack of tissue surface. Upper right: x/y-axis cross section. Lower left and right: x/z-axis cross sections. Blue, DNA; red, mCherry-MUC2; gray, actin. Data are means ± SEM (n = 3 or 4); *P < 0.05, Tukey multiple-comparisons test; ns, not significant. Scale bars, 50 μm (A), 20 μm [(B) and (E)]. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS

Fig. 3 TLR ligand–driven Muc2 secretion requires endocytosis, signaling, and ROS synthesis upstream of inflammasome activation. TLR ligand–driven Muc2 secretion requires endocytosis, signaling, and ROS synthesis upstream of inflammasome activation. Colonic explants [(A) to (C)] or cell suspensions [(D) and (E)] were treated with TLR ligands or CCh. (A) Quantification of mucus growth in wild-type (WT) or KO explants. (B) Quantification of mucus growth in explants pretreated with inhibitors; IP, inhibitory peptide. (C) Quantification of mucus growth in explants pretreated with ROS scavengers. (D) DCFDA fluorescence in epithelial cells pretreated with inhibitors. (E) Confocal micrographs of RedMUC298trTg epithelial cells with caspase inhibitory peptide. Purple arrowheads, nonendocytotic GCs; yellow arrowheads, endocytotic GCs; red, mCherry-MUC2; blue, DNA; gray, actin. Data are means ± SEM (n = 4 or 5); *P < 0.05, Dunnett multiple-comparisons test of WT versus KO (A) or no inhibitor versus inhibitor [(B) to (D)]. Scale bars, 50 μm. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS

Fig. 4 TLR ligands are endocytosed by sentinel GCs (senGCs). TLR ligands are endocytosed by sentinel GCs (senGCs). In (A) to (D), colonic explants were treated as indicated, whole-mounted, and visualized by confocal microscopy; in (A) to (C), x/y-axis cross sections are at top; x/z-axis cross sections are in yellow boxes; blue, DNA; gray, actin. (A) RedMUC298trTg tissue; regions of x/z-axis cross sections magnified at bottom are indicated by white boxes. Green, LPS/P3CSK4; red, mCherry-MUC2. (B) WT with or without caspase inhibitory peptide, Tlr2−/−, and MyD88−/− tissue. Green, P3CSK4. (C) RedMUC298trTg tissue. Yellow arrowheads, endocytotic GCs; white arrowheads, nonendocytotic GCs; red, mCherry-MUC2; green, dextran. (D) WT colon cotreated with P3CSK4 and dextran. Yellow arrowheads, endocytotic GCs. (E) Quantification of endocytotic cells. Data are means ± SEM (n = 3 or 4); *P < 0.05, Tukey multiple-comparisons test. Scale bars, 20 μm. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS

Fig. 5 Activated senGCs are expelled from the epithelium and trigger gap junction– and Ca2+-dependent Muc2 secretion from responsive upper-crypt GCs. Activated senGCs are expelled from the epithelium and trigger gap junction– and Ca2+-dependent Muc2 secretion from responsive upper-crypt GCs. Colonic explants were treated with TLR ligands or CCh. (A and B) RedMUC298trTg tissue whole mounts visualized by confocal microscopy. Blue, DNA; gray, actin; red, mCherry-MUC2; green, LPS, P3CSK4, or dextran. (A) x/z-axis cross sections showing expelled senGCs (yellow arrowheads). (B) Expelled senGC overview (left) and isosurface enhanced view (right, yellow box). (C) Localization of inflammasome activity after LPS treatment of WT, KO, and RedMUC298trTg tissue. Green, caspase-1/caspase-11 fluorogenic probe (Casp1/11 FP); blue, DNA; yellow arrowheads, senGCs. (D to F) RedMUC298trTg tissue imaged by confocal microscopy. (D) x/y-axis view (top) and x/z-axis view (bottom) of a crypt at different time points after LPS treatment. Green arrowheads and green dashed lines, senGC; yellow arrowheads, responsive GC; blue arrowheads, nonresponsive GC. (E) Quantification of mCherry-MUC2 fluorescence of individual GCs from crypt in (D). (F) Fluorescent isosurfaces used to generate data in (E); responsive GC numbering corresponds to numbered plots in (E). Red arrow shows sequence of responsive cell secretion. White, DNA; green, senGC; orange, responsive GCs; blue, nonresponsive GCs. (G and H) Quantification of mucus growth with or without CBX, TTx, or Ca2+ signaling inhibitors. (I and J) RedMUC298trTg tissue loaded with fluorogenic Ca2+ indicator (Fluo4) and imaged by confocal microscopy. Green, Fluo4; red, mCherry-MUC2. (I) x/y-axis view of tissue before and after treatment. (J) x/y-axis view of a crypt at different time points after LPS treatment. White arrow shows sequence of GC secretion. White dashed line, crypt opening; green arrowheads, senGC; yellow arrowheads, responsive GCs. Data are means ± SEM (n = 4 or 5); *P < 0.05, Dunnett multiple-comparisons test. Scale bars, 20 μm. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS

Fig. 6 senGC activation removes bacteria from crypt openings ex vivo and is triggered by disruption of the inner colonic mucus layer in vivo. senGC activation removes bacteria from crypt openings ex vivo and is triggered by disruption of the inner colonic mucus layer in vivo. (A) Fluorescent bacteria (red) applied to colonic explant tissue (green) and imaged before (top) and after (bottom) LPS or vehicle treatment. Left, x/z-axis view; right, x/y-axis view. Yellow arrowheads and dashed circles denote crypt openings. (B) Distribution of bacteria in relation to crypt openings as in (A). Data are representative of four independent experiments. In (C) to (F), mice were given 3% DSS in drinking water; samples were collected after 0 (no DSS), 12, 36, and 84 hours. (C) Confocal z-stacks of colonic explants and 1-μm beads. Blue, tissue; red, beads; im, impenetrable mucus. (D) Quantification of cells with inflammasome activity (Casp1/11 FP+) detected in live tissue. (E) Casp1/11 FP+ cells (pink arrows) with GC morphology in the upper crypt (left) and shed from tissue (right). Top, x/y-axis view; bottom, x/z-axis view. (F) Colonic explants were treated with fluorescent dextran, fixed, whole-mounted, and visualized by confocal microscopy. Top, x/y-axis view; bottom, x/z-axis view. Blue, DNA; green, dextran; gray, actin. Data are means ± SEM (n = 4); *P < 0.05, Dunn multiple-comparisons test. Scale bars, 20 μm. George M. H. Birchenough et al. Science 2016;352:1535-1542 Published by AAAS