The effect of age on the intestinal mucus thickness, microbiota composition and immunity in relation to sex in mice

A mucus layer covers and protects the intestinal epithelial cells from direct contact with microbes. This mucus layer not only prevents inflammation but also plays an essential role in microbiota colonization, indicating the complex interplay between mucus composition-microbiota and intestinal health. However, it is unknown whether the mucus layer is influenced by age or sex and whether this contributes to reported differences in intestinal diseases in males and females or with ageing. Therefore, in this study we investigated the effect of age on mucus thickness, intestinal microbiota composition and immune composition in relation to sex. The ageing induced shrinkage of the colonic mucus layer was associated with bacterial penetration and direct contact of bacteria with the epithelium in both sexes. Additionally, several genes involved in the biosynthesis of mucus were downregulated in old mice, especially in males, and this was accompanied by a decrease in abundances of various Lactobacillus species and unclassified Clostridiales type IV and XIV and increase in abundance of the potential pathobiont Bacteroides vulgatus. The changes in mucus and microbiota in old mice were associated with enhanced activation of the immune system as illustrated by a higher percentage of effector T cells in old mice. Our data contribute to a better understanding of the interplay between mucus-microbiota-and immune responses and ultimately may lead to more tailored design of strategies to modulate mucus production in targeted groups.</p

Aging-induced decline in mucus thickness in mice is associated with changes in microbiota composition and immunity and is sex dependent

A mucus layer covers and protects the intestinal epithelial cells from direct contact with microbes. This mucus layer not only prevents inflammation but also plays an essential role in microbiota colonization, indicating the complex interplay between mucus composition-microbiota and intestinal health. However, it is unknown whether the mucus layer is influenced by age or sex and whether this contributes to reported differences in intestinal diseases in males and females or with ageing. Therefore, in this study we investigated the effect of age on mucus thickness, intestinal microbiota composition and immune composition in relation to sex. The ageing induced shrinkage of the colonic mucus layer was associated with bacterial penetration and direct contact of bacteria with the epithelium in both sexes. Additionally, several genes involved in the biosynthesis of mucus were downregulated in old mice, especially in males, and this was accompanied by a decrease in abundances of various Lactobacillus species and unclassified Clostridiales type IV and XIV and increase in abundance of the potential pathobiont Bacteroides vulgatus. The changes in mucus and microbiota in old mice were associated with enhanced activation of the immune system as illustrated by a higher percentage of effector T cells in old mice. Our data contribute to a better understanding of the interplay between mucus-microbiota-and immune responses and ultimately may lead to more tailored design of strategies to modulate mucus production in targeted groups

Effect of age and sex on T lymphocytes in the Peyer’s patches.

<p>Percentage of CD3⁺ T lymphocytes (A), the percentage of T helper cells (CD4⁺) (B), T cytotoxic cells (CD8⁺) (C), and the percentage of expression of CD69⁺CD4⁺ (D) and CD69⁺CD8⁺ (E) in the Peyer’s patches (PP) of young (3 months) and old (19 months) male and female B6 mice. T helper and T cytotoxic cells are expressed as the frequency of CD4⁺ and CD8⁺ cells within the CD3⁺ population respectively. Results are expressed as dot plots + means and were tested using a Two-way ANOVA for overall age and sex effects, followed by a Bonferroni post-test for comparison between groups. Significant age effects are indicated with dashed lines and significant sex effects are indicated with solid lines (p<0.05). An additional group of ovariectomized (ovx) old females was added and compared with the old females with a t-test to determine the effect of a loss of female sex hormones (human menopause).</p

Effect of age and sex on T cell differentiation in the spleens.

<p>Percentage of CD62L⁺CD44^- naive CD4⁺ (A) and CD8⁺ (D), CD62L⁺CD44⁺ central memory CD4⁺ (B) and CD8⁺ (E), CD62L^-CD44⁺ effector memory CD4⁺ (C) and CD8⁺ (F), T helper 1 (G), T helper 2 (H), T helper 17 (I), FoxP3⁺CD25^- T regulatory cells (J), and FoxP3⁺CD25⁺ T regulatory cells (K) in the spleen of young (3 months) and old (19 months) male and female B6 mice. T helper and T cytotoxic cells are expressed as the frequency of CD4⁺ and CD8⁺ cells within the CD3⁺ population respectively. Results are expressed as dot plots + means and were tested using Two-way ANOVA, followed by a Bonferroni post-test for comparison between groups. Significant age effects are indicated with dashed lines and significant sex effects are indicated with solid lines (p<0.05). An additional group of ovariectomized (ovx) old females was added and compared with the old females with a t-test to determine the effect of a loss of female sex hormones (human menopause).</p

The effect of age on the abundance of several bacteria groups is sex specific.

<p>Heat-maps showing the abundance of several bacteria that differed significantly between young (3 months) and old (19 months) males and females. A box indicates bacteria which have a significantly different abundance between the two groups within that box. Colors indicate relative abundances normalized per bacterial group (per row), dark blue is the lowest abundance and dark red the highest abundance detected over all the samples of a bacterial group.</p

Effect of age and sex on T cell differentiation in the Peyer’s patches.

<p>Percentage of CD62L⁺CD44^- naive CD4⁺ (A) and CD8⁺ (D), CD62L⁺CD44⁺ central memory CD4⁺ (B) and CD8⁺ (E), CD62L^-CD44⁺ effector memory CD4⁺ (C) and CD8⁺ (F), T helper 1 (G), T helper 2 (H), T helper 17 (I), FoxP3⁺CD25^- T regulatory cells (J), and FoxP3⁺CD25⁺ T regulatory cells (K) in the Peyer’s patches (PP) of young (3 months) and old (19 months) male and female B6 mice. T helper and T cytotoxic cells are expressed as the frequency of CD4⁺ and CD8⁺ cells within the CD3⁺ population respectively. Results are expressed as dot plots + means and were tested using Two-way ANOVA, followed by a Bonferroni post-test for comparison between groups. Significant age effects are indicated with dashed lines and significant sex effects are indicated with solid lines (p<0.05). An additional group of ovariectomized (ovx) old females was added and compared with the old females with a t-test to determine the effect of a loss of female sex hormones (human menopause).</p

Effect of age and sex on fecal microbiota composition.

<p>Graphs showing the Shannon diversity (A) and the richness (C) in the fecal microbiota of male and female and young (3 months) and old (19 months) B6 mice. In addition, we collected feces from the old mice at three previous ages (8, 13 and 15 months). The Shannon diversity (B) and richness (D) of these time points, including the data of the young mice (3 months) are shown. Results are expressed as dot plots + means and were tested using a Two-way ANOVA for overall age and sex effects, followed by a Bonferroni post-test for comparison between groups. An additional group of ovariectomized (ovx) old females was added and compared with the old females to determine the effect of a loss of female sex hormones (human menopause). Significant effects are indicated with an asterisk (*) (p<0.05), while a trend (0.1</p><p>0.05) is indicated with a hash (#).</p

Effect of age and sex on T lymphocytes in the spleen.

<p>Percentage of CD3⁺ T lymphocytes (A), the percentage of T helper cells (CD4⁺) (B), T cytotoxic cells (CD8⁺) (C), and the percentage of expression of CD69⁺CD4⁺ (D) and CD69⁺CD8⁺ (E) in the spleen of young (3 months) and old (19 months) male and female B6 mice. T helper and T cytotoxic cells are expressed as the frequency of CD4⁺ and CD8⁺ cells within the CD3⁺ population respectively. Results are expressed as dot plots + means and were tested using Two-way ANOVA, followed by a Bonferroni post-test for comparison between groups. Significant age effects are indicated with dashed lines and significant sex effects are indicated with solid lines (p<0.05). An additional group of ovariectomized (ovx) old females was added and compared with the old females with a t-test to determine the effect of a loss of female sex hormones (human menopause).</p

The effect of age and sex on the mucus thickness in the colon.

<p>Representative pictures of PAS/Alcian Blue staining of the colon of young (Y) (3 months), old male (MO) (19 months), old female (FO) (19 months) and old ovariectomized female (FOvx) (19 months) B6 mice. Since young male and female mice showed a similar mucus layer morphology, only one representative image is shown for young mice. The mucus is indicated in blue and black arrows. Scale bars: 100 μm (A). Representative pictures of FISH staining of the colon of young, old male, old female and old ovx female mice, using the general bacterial probe EUB338-Alexa Fluor 488 (green), and nuclear staining DRAQ5 (blue). The apical membranes of the epithelial cells are indicated by a dashed white line. Arrow represents the gap between bacteria and epithelium in young healthy colon. This gap is not observed in colon from old mice, and the bacteria are close to the epithelium. Scale bars: 50 μm (B). Mucus thickness measured in PAS/Alcian Blue stained sections (10 measurements per section and 2 sections per mouse) in 5 colonic tissues of young and old mice. Significant effects are indicated with an asterisk (*) (Mann-Whitney U test, p<0.05). An additional group of ovariectomized (ovx) old females was added and compared with the old females to determine the effect of a loss of female sex hormones (human menopause) (C).</p