Fig 1.
The effect of age and sex on the mucus thickness in the colon.
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).
Fig 2.
Effect of age and sex on fecal microbiota composition.
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>0.05) is indicated with a hash (#).
Fig 3.
The effect of age on the abundance of several bacteria groups is sex specific.
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.
Fig 4.
Effect of age and sex on T lymphocytes in the Peyer’s patches.
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).
Fig 5.
Effect of age and sex on T lymphocytes in the spleen.
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).
Fig 6.
Effect of age and sex on T cell differentiation in the Peyer’s patches.
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).
Fig 7.
Effect of age and sex on T cell differentiation in the spleens.
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).
Fig 8.
Effect of age and sex on the percentage of dendritic cells in the Peyer’s patches.
Percentage of MHC2+CD64-CD19-CD11c+ dendritic cells (DCs) (A) and their expression of CD103 (B) in the Peyer’s patches (PP) of young (3 months) and old (19 months) male and female B6 mice. First leukocytes cells were selected based on size in the forward side scatter plot. DCs are expressed as the percentage MHC2+CD64-CD19-CD11c+ cells within all leukocytes. 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).