Fig 2: PMNFs ameliorates intestinal barrier breakdown induced by DSS in the colon of mice. (a) IHC staining of MUC-2 in the colon and the quantification of MUC-2. Scale bars, 50 µm. (b) Representative image of goblet cells in the colon as determined by PAS staining and the quantification numbers of goblet cells. Scale bars, 50 µm. (c) Representative image of apoptosis in the colon as determined by TUNEL staining and the quantification numbers of apoptotic cells per group. Arrows denote cells undergoing apoptosis. Scale bars, 50 µm. (d) Western blot analysis of claudin-1, occludin and ZO-1 and the quantification data in the colon. (e) IHC staining and the quantification of claudin-1, occludin and ZO-1 in the colon. Scale bars, 50 µm. (f) The mRNA expression of claudin-1, occludin and ZO-1 in the colon, detected by qRT-PCR. For all quantifications, data (mean ± SD) were from the indicated number of independent experiments and analyzed with two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus DSS-induced colitis group.

Fig 3: Aged distal colon shows impaired mucus barrier integrity and low level of T-synthase. (a) FISH using EUB338 probe and MUC2 antibody on distal colonic sections of mice showing bacterial penetration into the inner mucus layer. The magnified images in the 2- and 24-mo-old mouse groups showing boxed regions. The magnified image in the 16-mo-old mouse group revealing bacterial penetration of the only 16-mo-old mouse. (b) Representative fluorescent images showing UEA1 staining in the distal colons of 2-, 16-, and 24-mo-old mice. (c) Immunofluorescence staining of 2-, 16-, and 24-mo-old mouse colon sections with anti-Tn. (d) Western blot analysis showing the high expression of Tn antigen and sTn antigen in the old mouse distal mucosa compared to young. (e) T-synthase activity in the mouse distal colon and human colon. (f, g) Relative mRNA expression of C1GALT1 (f) and COSMC (g) and Western blot analysis of T-synthase (f) and Cosmc (g) protein expression in the mouse and human colon. Data are presented as mean ± SD. *** p < 0.001 compared with 2-mo-old mouse group or young people group; ## p < 0.01, ### p < 0.001 compared with 16-mo-old mouse group. Student's t test for (e-g Ms PC and Hu), and ANOVA and LSD test for (e-g Ms DC). n = 8 mice or 7 human samples per group. Abbreviations: Ms, mouse; Hu, human; PC, proximal colon; DC, distal colon.

Fig 4: Aged colons exhibit microbiota dysbiosis and a thinner mucus layer. (a) Real-time PCR analysis of pathogenic E. coli detected in the MLNs and liver of 24-mo-old mice but not 2-mo-old mice. (b–d) Relative mRNA fold changes or levels of TNF-a, IL-1ß, and IL-6 in young and old mouse livers (b) and the distal colonic mucosa of mice (c) and humans (d). (e) Relative fold changes of common bacteria in the distal colonic mucosa in old mice compared to young mice (standardized to 18S rRNA). (f) Representative H&E staining images of the distal colon from 2- and 24-mo-old mice. (g, h) Images showing AB/PAS staining (g) and MUC2 immunofluorescence staining (h) in the distal colons of young and old mice. Data are presented as mean ± SD. ***p < 0.001 compared with 2-mo-old mouse group or young people group. Student's t test or Mann–Whitney U test for (b–d). n = 8 mice or 7 human samples per group. Abbreviations: Ms, mouse; Hu, human.

Fig 5: Defects in mucus layer in UC colonoids are not due to decreased expression of DRA.(A) Changes in TEER of colonoid monolayers from HS (black triangle), and active UC (blue triangle) infected with adevovirus-3xflag-DRA HS (grey circle) and active UC (blue square). (B) Methanol–Carnoy’s fixed differentiated colonoid monolayers from HS and UC active stained with MUC2 (green), nucleus (blue). Representative confocal XZ (above) and 3D-XYZ (below) projections depicting the MUC2 layer in colonoids monolayer are shown. (C) Protein lysate prepared from colonoid monolayers uninfected or infected with 3x-flag-DRA was subjected to Western blot analysis and probed with DRA for endogenous expression and flag for overexpressed DRA. Representative results from 3 independent experiments with similar results are shown. Results are shown as mean ± SEM. *P < .05 vs HS; #P < .05 vs UC control.