Postnatal IML maturation is driven by both microbiota-dependent and -independent factors. (A) Total and taxon-specific colonic bacterial load in fetal and postnatal Wistar rats (left) and C57BL/6 mice (right) quantified by 16S qPCR of stool DNA. IML formation period between P2–P3 in rats is indicated. (B) Confocal micrographs of fixed mouse colonic tissue stained for luminal bacteria by 16S FISH. (C) Confocal micrographs of fixed colonic tissue sections stained for DNA (grey) and Muc2 (green). Dashed lines in high-magnification images (purple boxes) show tissue surface (orange) and IML border (yellow). Stratified Muc2 layers present in ConvR but not GF P3 IML are indicated (arrowheads) in highest magnification images (red boxes). (D) Comparison of gene expression ratios between ConvR P14:P3 mice (age dependent) and P14 ConvR:GF mice (microbiota dependent) quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly regulated by age (orange) or by both age and colonization status (blue) are indicated. (E) Ex vivo mucus growth in GF, ConvD, or ConvR tissue before (pre) and after (post) serine/cysteine protease inhibition by Comp PIC or the cysteine protease-specific inhibitor E64. (F) Ex vivo analysis of MyD88+/+ and MyD88−/− IML thickness and barrier function by microbead penetration. Images are x/z-axis cross-sections of confocal z-stacks showing colonic tissues (grey) overlaid with microbeads (red). Impenetrable mucus is indicated (yellow arrows). (G) Ex vivo analysis of MyD88+/+ and MyD88−/− IML thickness based on images shown in F. (H) Ex vivo analysis of MyD88+/+ and MyD88−/− IML barrier function based on images shown in F. (I) Ex vivo mucus growth in MyD88+/+ and MyD88−/− tissue in response to metalloprotease (EDTA) or serine/cysteine protease (Comp PIC) inhibition. Data represent n = 4–5 (A–C, E, and I), n = 6–8 (F–H), or n = 3 (D) animals per group, as indicated. All data are pooled from at least two independent litters or experiments. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by DESeq2 (D), Kruskal–Wallis and uncorrected Dunn’s test (E), or Mann–Whitney test (G–I); P < 0.05 (*), <0.01 (**). Image scale bars are 50 µm (B) or 100 µm (C). AUC, area under the curve.
Figure 2.

Postnatal IML maturation is driven by both microbiota-dependent and -independent factors. (A) Total and taxon-specific colonic bacterial load in fetal and postnatal Wistar rats (left) and C57BL/6 mice (right) quantified by 16S qPCR of stool DNA. IML formation period between P2–P3 in rats is indicated. (B) Confocal micrographs of fixed mouse colonic tissue stained for luminal bacteria by 16S FISH. (C) Confocal micrographs of fixed colonic tissue sections stained for DNA (grey) and Muc2 (green). Dashed lines in high-magnification images (purple boxes) show tissue surface (orange) and IML border (yellow). Stratified Muc2 layers present in ConvR but not GF P3 IML are indicated (arrowheads) in highest magnification images (red boxes). (D) Comparison of gene expression ratios between ConvR P14:P3 mice (age dependent) and P14 ConvR:GF mice (microbiota dependent) quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly regulated by age (orange) or by both age and colonization status (blue) are indicated. (E) Ex vivo mucus growth in GF, ConvD, or ConvR tissue before (pre) and after (post) serine/cysteine protease inhibition by Comp PIC or the cysteine protease-specific inhibitor E64. (F) Ex vivo analysis of MyD88+/+ and MyD88−/− IML thickness and barrier function by microbead penetration. Images are x/z-axis cross-sections of confocal z-stacks showing colonic tissues (grey) overlaid with microbeads (red). Impenetrable mucus is indicated (yellow arrows). (G) Ex vivo analysis of MyD88+/+ and MyD88−/− IML thickness based on images shown in F. (H) Ex vivo analysis of MyD88+/+ and MyD88−/− IML barrier function based on images shown in F. (I) Ex vivo mucus growth in MyD88+/+ and MyD88−/− tissue in response to metalloprotease (EDTA) or serine/cysteine protease (Comp PIC) inhibition. Data represent n = 4–5 (A–C, E, and I), n = 6–8 (F–H), or n = 3 (D) animals per group, as indicated. All data are pooled from at least two independent litters or experiments. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by DESeq2 (D), Kruskal–Wallis and uncorrected Dunn’s test (E), or Mann–Whitney test (G–I); P < 0.05 (*), <0.01 (**). Image scale bars are 50 µm (B) or 100 µm (C). AUC, area under the curve.

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