Gut microbial communities in young and aged mice

We began our investigation by examining the gut microbiota from mice at different ages. Cecal samples were collected from mice maintained for 8 weeks on a normal (low-fat) diet to investigate changes in the gut microbiota by pyrosequencing. The number of sequences analyzed, OTUs, estimated OTU richness (ACE and Chao1), and pyrosequencing coverage were calculated using Cluster Database at High Identity with Tolerance (Table 1). Decreased bacterial richness and diversity were observed in cecal samples collected from aged mice relative to those from young mice; however, these differences are not statistically significant.

Table 1 Number of sequences analyzed, observed diversity richness (OTU), estimated OTU richness (ACE and Chao1), and coverage Full size table

Taxonomy-based analysis showed that aging significantly modulated the populations of the dominant intestinal microbiota. At the phylum level, decreased levels of Bifidobacteria, which have been shown to exhibit anti-inflammatory effects in mice, and increased levels of Lactobacilli and Enterococci have been reported in elderly individuals compared with young adults [4]. In the present study, aging increased the prevalence of Firmicutes (p = 0.002) and Actinobacteria (p = 0.034) and reduced the prevalence of Bacteroidetes (p = 0.003) and Tenericutes (p = 0.038) (Fig. 1a,b), leading to an overall increase in the Firmicutes to Bacteroidetes ratio (p = 0.002) in the gut microbiota (Fig. 1c).

Fig. 1 The composition of intestinal microbiota. The relative contributions of dominant (a) phyla and (b) families (individual samples are shown in the left panels and pooled samples are shown in the right panels) and (c) the Firmicutes to Bacteroidetes ratio are shown as identified from pyrosequencing data. d Hierarchical clustering of gut microbial gene expression profiles. The distances between microbial communities from each sample are represented as an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering tree describing the dissimilarity between multiple samples. All values are indicated as the mean ± standard error of the mean (n = 4). *, p < 0.05 in comparison with young mice. YM, young mice; AM, aged mice Full size image

At the family level, Allobaculum_f (p = 0.002) and Clostridiaceae (p = 0.039) populations decreased, while Clostridiales_uc (p = 0.04) and EF602759_f (p = 0.008, phylum Bacteroidetes) populations were enriched in aged mice compared with young mice (Fig. 1b).

At the genus level, there were seven predominant genera. Six genera were in the phylum Firmicutes and one genus was in the phylum Bacteroidetes: DQ789121_g (phylum Firmicutes), Pseudoflavonifractor, EF603943_g (phylum Firmicutes), Bacteroides, EF603662_g (phylum Firmicutes), AB626958_g (phylum Firmicutes), and Clostridium_g9. Collectively, these seven genera accounted for more than 50 % of the sequences (Table 2). Among the 25 most abundant genera, EF603943_g, Clostridium_g9, and DQ815556_g of the phylum Firmicutes were enriched, while AY239469_g and EF406712_g of the phylum Bacteroidetes were reduced in aged mice in comparison with young mice.

Table 2 Difference in the composition (percent of total sequences) of fecal bacterial genera isolated from young mice and aged mice Full size table

We also analyzed the differences in the gut microbiota composition at the species level (Table 3). EF603943_s (Erysipelotrichales), FJ881142_s (Clostridiales), AY991787_s (Clostridiales), DQ815350_s (Clostridiales), EF098042_s (Clostridiales), AB606316_s AB606316_s (Clostridiales) of the phylum Firmicutes were enriched, while EF406712_s (Bacteroidales) and EF406368_s (Bacteroidales) of the phylum Bacteroidetes were reduced in aged mice in comparison with young mice.

Table 3 Difference in the composition (percent of total sequences) of fecal bacterial species isolated from young mice and aged mice Full size table

As shown in Fig. 1d, the samples from different groups could be readily separated, and four samples from each group clustered together.

Plasma and fecal endotoxin levels in young and aged mice

Inflammatory markers such as CD14, which is a LPS-binding protein in TLR4-linkged NF-κB signaling pathway, and C-reactive protein increase with advancing ages [17]. A previously unconsidered source of inflammatory initiation is the translocation of gut microbiota and their products, leading to inflammation such as inflammatory bowel disease.

To understand the role of LPS on the aging in mice, we measured the LPS levels in young and aged mice. We detected a significant increase (p < 0.05) in the epididymal fat pad weight/body weight ratio in aged mice as compared to young mice although there were no significant differences in the body weight gain (Fig. 2a).

Fig. 2 Effects of aging on endotoxin levels in young and aged mice. a Body weight (g) and epididymal fat pad weight (mg/g of body weight) of male C57BL/6 J mice (4 and 18 months old) were measured. b The fecal endotoxin concentration per gram (EU/g of feces) and plasma endotoxin concentration per mL (EU/mL) were measured using the Limulus amebocyte lysate assay. All values are indicated as the mean ± standard error of the mean (n = 8). *, p < 0.05 in comparison with young mice. YM, young mice; AM, aged mice Full size image

Next, we evaluated the endotoxin levels in the different treatment groups to investigate whether changes in gut microbiota composition with age are correlated with systemic endotoxemia. As shown in Fig. 2b, the fecal endotoxin levels in aged mice were higher than those in young mice. We also detected significantly higher systemic endotoxin levels in aged mice as compared to young mice.

Expression levels of p16, cell cycle regulators, and SAMHD1 in young and aged mice

Next, we measured the levels of p16, a senescence marker [6, 7], and cyclin E and CDK2, the G1/S-specific cell cycle regulators [18]. As indicated in Fig. 3, the expression of p16 was higher, while the expression levels of cyclin E and CDK2 were suppressed in aged mice relative to that in young mice. We also observed that activation of NF-κB and mTOR were stronger in aged mice than in young mice. Interestingly, concurrent with the increased expression of p16, the colonic expression of SAMHD1 was increased in aged mice relative to that in young mice.

Fig. 3 Effects of aging on inflammation, p16, cell cycle-regulators, and SAMHD1 levels in the colon of young and aged mice. Western blot analysis was performed on colon lysates from young mice or aged mice. All values are indicated as the mean ± standard error of the mean (n = 8). YM, young mice; AM, aged mice Full size image

To investigate whether higher levels of lipopolysaccharide (LPS) may trigger increased expression of p16 and SAMHD1, we treated peritoneal macrophages isolated from young mice with LPS ex vivo. We found that LPS increased the expression of p16 and SAMHD1 (Fig. 4).

Fig. 4 Effects of LPS on SAMHD1 expression in peritoneal macrophages. Peritoneal macrophages from mice were incubated with 10, 50, or 100 ng/mL lipopolysaccharides and used for immunoblotting. All values are indicated as the mean ± standard error of the mean (n = 3). YM, young mice; AM, aged mice Full size image

LFL increased SAMHD1 expression and inflammation in isolated macrophages

To investigate whether intestinal bacterial endotoxin could induce p16 and SAMHD1 expression, we isolated peritoneal macrophages from wild-type (WT) and TLR4-deficient mice and incubated them with fecal lysates from young and aged mice. The LFLs from aged mice induced greater p16 and SAMHD1 expression in peritoneal macrophages from WT mice than did those from young mice when the isolated peritoneal macrophages were treated with different concentrations of LPS (Fig. 5 top). However, when peritoneal macrophages from TLR4-deficient mice were incubated with LFLs, they slightly but not significantly increased NF-κB activation and SAMHD1 expression, whereas they significantly induced p16 expression (Fig. 5 bottom). Nevertheless, p16 expression was induced more potently in macrophages from WT mice than in macrophages from TLR4-deficient mice. Moreover, the difference of p16 and SAMHD1 expression NF-κB activation between young and aged mice was not significant.