Influencing mucus to change the microbiota

Our hypothesis is that HIF-1a expression influences mucus thickness, sterility, and glycan profile. <br><br>In this study, we used colonic epithelial cells in vitro; Caco2 (enterocyte cell line) and HT29MTX (goblet cell line). Cell lines were cultured either in normoxia, hypoxia or treated with pharmacological modulators of HIF to study the regulation of the mucin glycosyltransferase enzymes, mucus composition, and effect of altered mucus layer on commensals/pathogenesis bacteria growth.

Using differentiated Caco2, we showed higher C1GALT1 (the main mucin glycosylation enzyme) expression in hypoxia compared to normoxia over 8-hours of exposure time. We also studied the metabolically incorporation of different labelled sugars in mucin structure using “click chemistry” reaction. Azido labelled N-acetylgalactosamine (GalNAc) shows lower GalNAc incorporation in HIF inhibited mucus produced in goblet cells compare to HIF stabilised one.

N-acetylglucosamine (GlcNAc) revealed high incorporation in HMW mucus glycoprotein structures produced by the cells under Normoxia compared with mucus produced under hypoxic conditions. Mucin-2 Immunofluorescent labelling shows the higher MUC2 expression in HIF stabilised cells and not significant changes in normoxic and HIF inhibited cells which shows the role of HIF in adaptive immune respond in hypoxia. The antimicrobial activity of mucus secreted by HT29 and Caco2 was also studied using E. coli and S. Typhimurium as candidate for commensal and pathogenic bacteria, respectively.

The E. coli was sensitive to mucus produced by normoxic, hypoxic, and HIF inhibited cells. However, the S. Typhimurium showed not significant sensitivity to all mucus samples (a source of antimicrobial factor). Finally, to study the carbon source availability of mucus layer secreted by goblet cell line, in vitro, we used Akkermansia muciniphila, an anaerobic commensal mucin-degrading bacterial species, and incubated with different mucus samples (collected from differently treated cells) in minimal growth conditions (M9 salts) under anaerobic conditions and analysed its growth after 24 hours.

Subsequently, we studied the influence of liberated/residual glycans on the growth of commensal E. coli (K12 MG1655). Overall, the E. coli growth curve showed the bacteria supplemented with all the different mucus samples had more growth than the bacteria treated with only M9 salt media. The mucus sample produced under hypoxia and HIF stabilisation also provided higher growth for commensal bacteria compared with the bacteria treated with mucus sample produced under normoxic/HIF inhibitor conditions.

In conclusion, our findings indicate that the gut mucus properties are influenced by epithelial oxygenation state, and this may have a high influence on the composition of the microbiota. <br>