Age-associated Impairment of the Mucus Barrier Function is Associated with Profound Changes in Microbiota and Immunity

Aging significantly increases the vulnerability to gastrointestinal (GI) disorders but there are few studies investigating the key factors in aging that affect the GI tract. To address this knowledge gap, we used 10-week- and 19-month-old litter-mate mice to investigate microbiota and host gene expression changes in association with ageing. In aged mice the thickness of the colonic mucus layer was reduced about 6-fold relative to young mice, and more easily penetrable by luminal bacteria. This was linked to increased apoptosis of goblet cells in the upper part of the crypts. The barrier function of the small intestinal mucus was also compromised and the microbiota were frequently observed in contact with the villus epithelium. Antimicrobial Paneth cell factors Ang4 and lysozyme were expressed in significantly reduced amounts. These barrier defects were accompanied by major changes in the faecal microbiota and significantly decreased abundance of Akkermansia muciniphila which is strongly and negatively affected by old age in humans. Transcriptomics revealed age-associated decreases in the expression of immunity and other genes in intestinal mucosal tissue, including decreased T cell-specific transcripts and T cell signalling pathways. The physiological and immunological changes we observed in the intestine in old age, could have major consequences beyond the gut.</p

Laterality defect of the heart in non-teleost fish

Dextrocardia is a rare congenital malformation in humans in which most of the heart mass is positioned in the right hemithorax rather than on the left. The heart itself may be normal and dextrocardia is sometimes diagnosed during non-related explorations. A few reports have documented atypical positions of the cardiac chambers in farmed teleost fish. Here, we report the casual finding of a left-right mirrored heart in an 85 cm long wild-caught spiny dogfish (Squalus acanthias) with several organ malformations. Macroscopic observations showed an outflow tract originating from the left side of the ventricular mass, rather than from the right. Internal inspection revealed the expected structures and a looped cavity. The inner curvature of the loop comprised a large trabeculation, the bulboventricular fold, as expected. The junction between the sinus venosus and the atrium appeared normal, only mirrored. MRI data acquired at 0.7 mm isotropic resolution and subsequent 3D-modeling revealed the atrioventricular canal was to the right of the bulboventricular fold, rather than on the left. Spurred by the finding of dextrocardia in the shark, we revisit our previously published material on farmed Adriatic sturgeon (Acipenser naccarii), a non-teleost bony fish. We found several alevins with inverted (left-loop) hearts, amounting to an approximate incidence of 1%-2%. Additionally, an adult sturgeon measuring 90 cm in length showed abnormal topology of the cardiac chambers, but normal position of the abdominal organs. In conclusion, left-right mirrored hearts, a setting that resembles human dextrocardia, can occur in both farmed and wild non-teleost fish.ACKNOWLEDGMENTS. The authors wish to thank A. Domezain, from the Sierra Nevada Fishery at Riofrío, Granada, Spain, for generous access to the sturgeon material. Jaco Hagoort's help in generating the 3D model is much appreciate

Age-associated Impairment of the Mucus Barrier Function is Associated with Profound Changes in Microbiota and Immunity.

Aging significantly increases the vulnerability to gastrointestinal (GI) disorders but there are few studies investigating the key factors in aging that affect the GI tract. To address this knowledge gap, we used 10-week- and 19-month-old litter-mate mice to investigate microbiota and host gene expression changes in association with ageing. In aged mice the thickness of the colonic mucus layer was reduced about 6-fold relative to young mice, and more easily penetrable by luminal bacteria. This was linked to increased apoptosis of goblet cells in the upper part of the crypts. The barrier function of the small intestinal mucus was also compromised and the microbiota were frequently observed in contact with the villus epithelium. Antimicrobial Paneth cell factors Ang4 and lysozyme were expressed in significantly reduced amounts. These barrier defects were accompanied by major changes in the faecal microbiota and significantly decreased abundance of Akkermansia muciniphila which is strongly and negatively affected by old age in humans. Transcriptomics revealed age-associated decreases in the expression of immunity and other genes in intestinal mucosal tissue, including decreased T cell-specific transcripts and T cell signalling pathways. The physiological and immunological changes we observed in the intestine in old age, could have major consequences beyond the gut

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 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

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 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