Figure 3. Old microbiota disrupts gut barrier functions by reducing mucin expression and goblet cells in recipient mouse gut.

(A) Differential abundance data on an intestinal epithelia-related gene array showing significantly reduced expression of mucin genes Muc2 and Muc6 and tight junction gene Zo1 and increased expression of inflammatory genes Il6 and Tnfa in the intestine (ileum) of mice receiving FMT from old mice compared with controls receiving young FMT. (B) Random forest analysis shows that Muc2 was most significantly affected by old FMT. The mean decrease in gini score respresents the importance of the variable in building the model; thus, the higher the value of the mean decrease in gini score, the higher the importance of the variable in the model. (C and D) Old FMT recipients have significantly fewer goblet cells (periodic acid–Schiff [PAS] staining; red arrows) in their intestinal villi than controls. (E) Fecal mucin content was also significantly lower in old FMT recipients than in controls. (F and G) Treatment with fecal conditioned media (FCM) made from the feces of old mice significantly reduced transepithelial electrical resistance (TEER) (F) and increased FITC-dextran permeability (G) in the monolayers of human HT29 cells compared with treatment with young FCM. (H–K) Muc2 and Muc6 expression was significantly reduced in goblet-like CMT93 cells (H) and enteroids (I) treated with old FCM; they resembled the intestines (ileum and colon) of old FMT recipient mice (J and K). All the values represent the mean of 5–10 animals or 3–4 independent replicates for each group in the cell and enteroid experiments repeated 2–3 times, and error bars represent the standard error of means. Statistical significance was determined using t test and/or ANOVA, as applicable, and P values *P < 0.05, **P < 0.01, and ***P < 0.001 are statistically significant.