Our study demonstrated increased longevity in mice following probiotic treatment with LKM512, possibly due to the suppression of chronic low-grade inflammation in the colon induced by higher PA levels. This indicates that ingestion of specific probiotics may be an easy approach for improving intestinal health and increasing lifespan. Further studies are required to clarify its effectiveness in humans.

We supplemented the diet of 10-month-old Crj:CD-1 female mice with LKM512 for 11 months, while the controls received no supplementation. Survival rates were compared using Kaplan–Meier survival curves. LKM512-treated mice survived significantly longer than controls (P<0.001); moreover, skin ulcers and tumors were more common in the control mice. We then analyzed inflammatory and intestinal conditions by measuring several markers using HPLC, ELISA, reverse transcription-quantitative PCR, and histological slices. LKM512 mice showed altered 16S rRNA gene expression of several predominant intestinal bacterial groups. The fecal concentrations of PAs, but not of short-chain fatty acids, were significantly higher in LKM512-treated mice (P<0.05). Colonic mucosal function was also better in LKM512 mice, with increased mucus secretion and better maintenance of tight junctions. Changes in gene expression levels were evaluated using the NimbleGen mouse DNA microarray. LKM512 administration also downregulated the expression of ageing-associated and inflammation-associated genes and gene expression levels in 21-month-old LKM512-treated mice resembled those in 10-month-old untreated (younger) mice.

Chronic low-grade inflammation is recognized as an important factor contributing to senescence and age-related diseases. In mammals, levels of polyamines (PAs) decrease during the ageing process; PAs are known to decrease systemic inflammation by inhibiting inflammatory cytokine synthesis in macrophages. Reductions in intestinal luminal PAs levels have been associated with intestinal barrier dysfunction. The probiotic strain Bifidobacterium animalis subsp. lactis LKM512 is known to increase intestinal luminal PA concentrations.

Competing interests: M. Matsumoto is an employee of and holds equity in Kyodo Milk Industry Co. Ltd. All of the other authors declare that they have no conflict of interest. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Funding: This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry by the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was also funded by Kyodo Milk Industry Co. Ltd. M. Matsumoto is an employee of Kyodo Milk Industry Co. Ltd. and had a role in study design, data analysis, preparation of the manuscript, and the decision to publish the manuscript.

On the basis of these data, we hypothesized that the use of probiotics such as LKM512 would increase mammalian longevity by suppressing chronic low-grade inflammation [27] . To test these hypotheses, we examined middle-aged (10-month-old) mice that were provided a standard diet supplemented by oral doses of LKM512 or control. After 6 months of treatment (when the mice were 16 month old), LKM512 mice showed increased longevity as compared to the control mice.

Polyamines (PAs), such as putrescine (PUT), spermidine (SPD), and spermine (SPM), are organic cations required for cell growth, cell differentiation, and for the synthesis of DNA, RNA, and proteins [14] . PAs are known to possess anti-inflammatory activity via the inhibition of inflammatory cytokine synthesis in macrophages [15] and the regulation of NFκB activation [16] , and are closely associated with the maintenance of the intestinal mucosal barrier function [17] . Furthermore, PAs function as ROS scavengers, acid tolerance factors, chemical chaperones, and positive regulators for the expression of stress response genes [18] ; PAs also have antimutagenic activity [19] . As exogenous PAs derived from meals are absorbed before they reach the lower parts of the intestine [20] , it has been suggested that the greatest amounts of the PAs in the lower parts of the intestine are synthesized by intestinal microbiota [21] . In mammals, body PA levels decrease during the ageing process [22] ; intestinal PA concentrations in the elderly are lower than those in healthy adults [23] , suggesting that these compounds may be linked to senescence and longevity.

At least 1,000 bacterial species have been found to inhabit the human intestine, and 10 14 individual bacterial cells of at least 160 different species inhabit each individual's intestine [6] , which is 10 times greater than the total number of somatic and germ cells in the human body [7] . Intestinal microbiota plays a fundamentally important role in health and disease [7] . In the healthy intestinal tract, the microbiota and the gut-associated immune system are assumed to share a fine and dynamic homeostatic equilibrium [8] . The chronic low-grade inflammation process may undermine this balance. Although there have been no studies demonstrating the relationship between intestinal luminal environment, chronic low-grade inflammation , and lifespan, we believe that lifespan can be extended by the inhibition of chronic low-grade inflammation via the control of intestinal microbiota for the following 4 reasons. First, the lifespan of germ-free mice is longer than that of conventional mice [9] ; second, intestinal microbiota undergoes alterations by ageing [10] , [11] ; third, intestinal microbiota stimulates host mucosal and systemic immunity [12] ; and lastly, ageing-associated deterioration in intestinal barrier functions may permit increased systemic absorption of intestinal luminal antigens [13] .

Many mechanisms have been shown to contribute to the process of senescence, such as telomere shortening in replicative cells, cumulative damage to DNA leading to genomic instability, oxidative damage to critical molecules by reactive oxygen species (ROS), and so on [2] . These mechanisms also comprise chronic low-grade inflammation, a major risk factor for ageing and age-related diseases, such as Alzheimer's disease and type II diabetes [3] , [4] . Furthermore, the prevention of chronic low-grade inflammation appears to be one of the most effective approaches for increasing longevity [5] .

(A and B) Concentrations of urinary haptoglobin (A) and serum TNF-α (B). (C) Colonic gene expression of Traf6 and Tnf. Data are expressed as the mean relative amount to mRNA of β-actin ± SEM. * P<0.05. (D) Microarray data scatter plots of the genes in the TNF-NFκB pathway. All genes (n = 25,631) are represented on scatter plots. The values for all the TNF-NFκB pathway genes represented on the array are highlighted in black. Younger (pretreatment) vs. control (left), LKM512 vs. control (middle), and LKM512 vs. younger mice (right).

We used urine haptoglobin levels to estimate intestinal inflammation [31] . At 25 weeks, haptoglobin levels were lower in LKM512 mice than in control mice (P<0.05) ( Fig. 5A ). Additionally, at 45 weeks, serum TNF-α concentrations tended to be lower in LKM512 mice than in control mice ( Fig. 5B ), as were gene expression levels of colonic traf6 and Tnf ( Fig. 5C ) (P<0.05). These results indicate that LKM512 administration suppressed systemic and colonic inflammation caused by ageing. The anti-inflammatory effects of LKM512 administration were also revealed by a DNA microarray. Expression levels of genes in the TNF-NFκB, IL-1, IL-2, and IL-6 pathways were higher in the control group than in LKM512 and younger mice; additionally, gene expression levels in LKM512 mice were similar to those in younger mice ( Fig. 5D and Fig. S3 ).

When the mice aged or were administered LKM512 treatments, 55 of 93 gene pathways were altered significantly (Z-score >1.98), while the gene expression for 78 of these 93 gene pathways was similar between younger mice and mice receiving LKM512 ( Fig. 4C , Fig. S2 ). Pathways that were downregulated by ageing were upregulated by LKM512 administration and vice versa. In other words, LKM512 administration suppressed ageing-associated change in gene pathways.

(A) Hierarchical clustering showing patterns of expression relationships among LKM512-treated, control, and younger mice. Red and green indicate up- and downregulation of gene expression, respectively. (B) The number of genes up- and downregulated during the ageing process in each of the putative functional categories on microarray analysis. (C) Comparison of pathways that were significantly upregulated (red) and downregulated (blue) by LKM512 administration and ageing.

We examined gene expression patterns in the middle colons of mice in each of the 3 groups, i.e., LKM512 mice, control, and younger (pretreatment) mice ( Fig. 4A ). Using 4-fold change as a cutoff, we detected 11,164 differentially expressed genes. Although expression patterns in LKM512 mice were similar to those in younger mice, these patterns contrasted with that of control mice. This suggests that senescence-associated colonic gene expression was suppressed by LKM512 administration. By combining the functional annotations for these genes, we were able to assign multiple biological functions to certain genes and classify them into 9 putative functional categories. This allowed us to determine that, in most of these categories, a greater number of genes were upregulated than downregulated during the ageing process ( Fig. 4B ).

LKM512 treatment changed the gene expression of some intestinal barrier associated proteins ( Fig. 3C ). Administration of LKM512 tended to increase the expression of MUC2, a mucin that is secreted by goblet cells to form the colonic mucus layer (P = 0.07). Levels of MUC2 gene expression in the LKM512 group were equivalent to those observed in younger mice. However, colonic gene expression of MUC3, a membrane-bound mucin, was not altered by LKM512 administration. Colonic gene expression of occludin, one of the tight junction-associated proteins that help maintain intestinal barrier function, was increased by LKM512 administration (P<0.05). However, zonula occludens (ZO)-1 was not altered by LKM512 administration.

(A) Histology of proximal colon samples from mice treated with LKM512 (left) or PBS (control; right). The samples shown in the upper panels are stained with HE, while those in the bottom panels are stained with PAS. [Scale bars, 500 µm (HE), 100 µm (PAS).] (B) Colon permeability at week 25, as indicated by the urinary lactulose/rhamnose ratio. (C) Colonic gene expression of Muc2, Muc3, Occludin, and ZO-1 in all treatment groups and in younger mice. Data are represented as mean ± SEM. *P<0.05.

Mice that received LKM512 treatments had functional colonic mucosal layers; for instance, goblet cells were observed along the length of the crypt. On the other hand, there were clear signs of degradation in mucosal function in control mice, in whom the crypts were degraded and few goblet cells were observed in ∼50% of the control mice that survived until week 45 (21 months) ( Fig. 3A ). At week 25, the urinary lactulose/rhamnose (L/R) ratio of LKM512 mice was significantly lower than that in control mice (P<0.05) ( Fig. 3B ). This indicated improved maintenance of the intestinal barrier function by LKM512 treatment [30] .

The fecal concentrations of PAs, particularly SPM, which is the most bioactive PA, were significantly higher in the LKM512 mice than in the control mice (P<0.05, Fig. 2C ). The correlation between fecal SPM concentration and 16S rRNA gene expression of the predominant bacterial group is shown in Fig. S1 . This may have been due to the activity of B. animalis subsp. lactis (LKM512) and/or Prevotella spp., caused by the administration of LKM512, since the SPM concentration correlated better with the 16S rRNA gene expression of these bacteria as compared to other bacterial groups ( Fig. S1 ). However, in this study, we could not specifically identify the bacterial group that produces PAs. Moreover, the complete genome sequence for B. animalis subsp. lactis indicates that this species does not possess the pathways for SPM synthesis; therefore, it was clear that LKM512 itself did not produce SPM. LKM512 administration did not significantly change short chain fatty acids (SCFA) concentrations ( Fig. 2D ). Fecal mutagenicity was significantly lower in the LKM512 mice than in control mice (P<0.05, Fig. 2E ). PAs are known to have strong bioantimutagenic activities [19] , we considered that this might be due to the increase in SPM concentration caused by LKM512 administration. However, other factors may have been involved in decreasing mutagenicity, e.g., binding of the mutagen to LKM512 cell walls as well as those of other bifidobacterial strains [29] or a decrease in mutagens caused by enzymes derived from the altered intestinal microbiota, such as β-glucuronidase.

We used reverse transcription-quantitative PCR (RT-qPCR)to quantify commensal gut bacteria by measuring their expression of the 16S rRNA gene. In general, we found that the LKM512 treatments altered 16S rRNA gene expression of several predominant bacterial groups by week 45 ( Fig. 2B ). Specifically, the 16S rRNA gene expression of both B. animalis subsp. lactis (LKM512) and Prevotella spp. were detected in all mice from the LKM512 group and were higher than that in control mice (P<0.01 and P<0.001, respectively). To the best of our knowledge, this is the first demonstration that Prevotella counts may be increased by the administration of probiotics. Furthermore, these values were higher at the end of the LKM512-treatment period when the mice were 21 months old, than before the treatment began when mice were 10 months old (younger mice) (P<0.001). Following LKM512 treatment, 16S rRNA gene expressions of Clostridium cluster XIVa (vs. younger mice, P<0.05) were also altered. We also found evidence that LKM512 administration suppressed age-related changes in intestinal microbiota: 16S rRNA gene expression of the Bacteroides fragilis group (P<0.05) and Enterobacteriaceae species (P = 0.06) differed between the control group and younger mice, but not between the LKM512-treated and younger mice. Other bacterial genera showed no alterations.

(A) Appearance of large intestine obtained from LKM512 (upper) and control (bottom) mice at week 45. (B) 16S rRNA gene expression in the predominant intestinal bacterial group at week 45. 16S rRNA gene expression in the bacterial group was normalized to total bacterial 16S rRNA expression. Expression in control and younger mice is shown relative to the expression in the LKM512 mice. (C) Fecal SPD (right) and SPM (left) concentrations in each group. (D) The concentration of SCFA in each group. (E) Fecal mutagenicity stimulated with S-9 mix and without S-9 mix in each group. In (B) – (E), data are represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

In the mice in the control group, the incidence of visible tumors (P<0.05) and skin ulcers (P = 0.155) was higher than that in the LKM512 treatment group ( Fig. 1C–E ). Additionally, skin and hair quality was better in LKM512 mice than in control mice, indicating that this treatment allows mice to not only live longer, but also be healthier as they age. These results were not influenced by the stress of establishing and maintaining social rank, because we observed no fighting amongst the female mice used in this study.

Mice treated with LKM512 lived significantly longer than controls (P<0.001) ( Fig. 1A ). The survival curves of the 2 groups began to diverge at 20 weeks (when the mice were 15 months old), and remained apart until the end of the study. However, there was no significant weight difference between the groups, or any noticeable difference in weight fluctuation during the study period, although mice in both treatment groups were given a standard pellet chow diet ad libitum ( Fig. 1B ). It is important to note that the longevity observed in the LKM512-treated mice was not related to calorie restriction (CR), which has been shown to increase longevity in a variety of species [28] .

Discussion

In the present study, we have shown that the lifespan of mammals can be increased by probiotic treatment; furthermore, we have proposed that the mechanism by which this longevity was achieved is the suppression of chronic low-grade inflammation resulting from improvements in the intestinal luminal environment and the maintenance of colon tissue.

It is generally believed that intestinal bacterial components are recognized by Toll-like receptors under normal steady-state conditions, and this interaction plays a crucial role in host immunity [33]. Therefore, there is a possibility that this anti-chronic low-grade inflammation effect depends on the immunostimulation of intestinal microbiota altered by LKM512 treatment. However, our previous study demonstrated that the anti-inflammatory benefits of LKM512 are influenced by intestinal bacterial metabolites more directly than by immunostimulation due to bacterial cell components derived from certain strains within an altered colonic microbiota [34], [35]. There is also a possibility that orally administrated LKM512 cells directly stimulate lymphocytes in the Peyer's patches when they pass through the small intestine, similar to the effect of Lactobacillus casei strain Shirota [36], irrespective of the colonic luminal environment. Although this may be one of the mechanisms by which chronic low-grade inflammation was suppressed by LKM512 treatment, it cannot explain the inhibition of colonic inflammatory gene expression or the maintenance of colonic barrier function. We focused upon the fact that intestinal microbiota produce metabolites that have been shown to influence inflammation; for example, levels of PAs, which possess anti-inflammatory activity [15], [16] and the ability to maintain intestinal mucosal barrier functions [17], [37], were increased by LKM512 treatment.

By focusing on PAs as principal bioactive substances, we proposed the hypothesis that increased intestinal levels of PAs would increase longevity by improving intestinal health and inhibiting systemic chronic low-grade inflammation (details in Fig. 6). Improved long-term survival has been demonstrated for SPD-treated cells and organisms [38], while PA-rich foods are known to decrease age-associated pathology and mortality in aged mice [39]. However, the oral administration of SPM did not improve longevity to the same extent as did the dose-intrinsic PAs supplied by colonic microbiota altered by LKM512 administration (P = 0.121) (Fig. S5A). This may be related to the quantity of PAs supplied: PAs delivered orally are transient, but PAs produced by colonic microbiota that have been enhanced by LKM512 treatment are continuously replaced. This allows for a much larger “dose” that is suitable for increasing longevity. This theory is supported by the differences in colonic SPM concentrations between LKM512 mice and SPM mice, wherein the SPM levels in SPM mice were as low as those in control mice (Fig. S5B), as well as by the differences in gene expression patterns (Fig. S5C). It is possible that other probiotic strains that have the ability to influence the intestinal environment, such as B. bifidum PRL2010—which metabolizes mucin derived from the host [40], and Lactobacillus acidophilus NCFM [41]—which increases intestinal PA levels following oral administration, may promote longevity. By using gnotobiotic mice singly associated with Escherichia coli O157, Fukuda et al. [42] demonstrated the protective abilities of Bifidobacterium logum JCM 1217T against enteropathogenic infections through the production of acetate. However, acetate concentrations were not altered by LKM512 administration (Fig. 2D), suggesting that the promotion of longevity by LKM512 is not related to the protection against pathogenic bacterial infections by the acetate produced by LKM512 or other bacteria.

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larger image TIFF original image Download: Figure 6. The hypothetical mechanism behind the increased lifespan of LKM512-treated mice (this is a modification of a part of a previous hypothesis The hypothetical mechanism behind the increased lifespan of LKM512-treated mice (this is a modification of a part of a previous hypothesis [27] ). After LKM512 is orally administered, it colonizes the colon and alters the intestinal microbiota, which then produces PAs. The alteration in intestinal microbiota by LKM512 facilitates the activity of Prevotella spp. but suppresses the Bact. fragilis group, Enterobacteriaceae species, and Enterococcus spp. The produced PAs induces maintenance and/or recovery of intestinal barrier function by upregulating mucous secretion; additionally, because of its antioxidative properties, it helps prevent colonic barrier disruption. Maintenance of the intestinal barrier reduces the intestinal inflammatory activity of factors derived from food and bacteria. Moreover, increased levels of PAs in the intestinal lumen lead to increased PA concentration in the blood. This circulating PA suppresses the induction and/or production of systemic inflammatory cytokines. At the same time, PAs possess bioantimutagenic and antioxidative activities that prevent DNA damage. Thus, the effects of PAs suppresschronic low-grade inflammation , thereby promoting longevity. Furthermore, although data is insufficient in this study, there is a possibility that autophagy induced by PAs also promotes longevity. https://doi.org/10.1371/journal.pone.0023652.g006

This study proposes a new finding concerning the relationship between the colon and antiaging. Cumulatively, treatment with LKM512 inhibited morphological disruption of the intestinal barrier during the ageing process [13], suggesting that maintenance of intestinal barrier function was one of the major factors in the promotion of longevity in this trial. Furthermore, LKM512 administration suppressed senescence of the colon by downregulating ageing-associated gene expression, rather than by upregulating rejuvenation-associated gene expression (Fig 4B). A report has associated modulation in gene expression through ageing with compromised intestinal function and propensity for colon cancer in the rat colon. We compared the changes in colonic gene expression in rats in that report [43] with our data (Table S1). In this study, we noted that almost all the genes upregulated by ageing in rats were similarly upregulated by ageing in mice; furthermore, these were downregulated by LKM512 treatment. It is interesting to note that the pathways (Fig. 4C; upper panel) that were upregulated by LKM512 administration and downregulated by ageing were G-protein-coupled receptor (GPCR)-related pathways. Currently, there is little information available regarding the relationship between GPCRs, intestinal microbiota, longevity, and/or PAs, although it is known that SCFA and GPCR interact to affect the inflammatory response [44]. Clearly, further studies are required to clarify these relationships. To the best of our knowledge, this is the first demonstration of anti-ageing in the colon by probiotics using global gene expression profiling. CR is known as one of the factors capable of increasing the lifespan in a variety of species [28]. CR-induced metabolic reprogramming may be a key event in the mechanism of lifespan extension [45]. Studies in yeast, worms, flies, and mice point to its influence on nutrient-responsive signaling molecules, including SIRT1, the mammalian target of rapamycin (mTOR), and proliferator-activated receptor-γ coactivator 1α (PGC-1α) [46]. On the other hand, lifespan extension has been demonstrated to be induced by the control of these signaling molecules even in the absence of CR, i.e., by the oral administration of rapamycin [47] and resveratrol [48]. Rapamycin reduces the expression of function of mTOR, which is a central regulator of many biological processes [49]. It is known that the genetic inhibition of TOR extends the lifespan in short-lived model organisms and mice [47]. In our present study, the mTOR pathway was not listed in the GenMAPP pathway analysis; however, since rapamycin suppresses the mTORC1 complex in the mTOR pathway, we analyzed genes downstream of mTORC1 (Table S2). Although 6 out of 10 genes were upregulated by ageing, 4 out of the 6 genes thus upregulated were downregulated by LKM512 treatment, indicating that one of the mechanisms of lifespan extension by LKM512 treatment may involve the suppression of the mTOR pathway, similar to the effect of rapamycin. Further research is warranted in order to confirm the phosphorylation levels of at least 2 substrates, i.e., P70-S6 kinase that phosphorylates S6 ribosomal protein and 4E binding protein 1, which is a key translational repressor protein.

Resveratrol, a pharmacological activator of SIRT1, can improve the lifespan and health of mice on a typical high-calorie diet [48]. Its effects lead to a decrease in the levels of insulin-like growth factor-1 (IGF-1), while AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) activity is increased [48]. However, in our present study, while LKM512 treatment caused only slight changes in IGF-1 gene expression, AMPK-related and PGC-1α gene expression were considerably decreased (Table S2), indicating that the mechanism underlying improved longevity by LKM512 treatment differed from resveratrol's mechanism. Recently, autophagy—the cellular process of cytoplasmic degradation and recycling—has been proposed to promote longevity [38]. Interestingly, all the above mentioned longevity-promoting regimens—including CR and inhibition of TOR with rapamycin, resveratrol, or PAs—have been associated with autophagy; in some cases, they have been reported to require autophagy for their effects [50]. In fact, colonic autophagy appeared to be promoted in LKM512 mice as compared to the controls, as revealed by LC3 conversion (Fig. S6).

At less than 36 weeks of treatment, the median survival of the control group was less than 19 months, which was considerably short in comparison to those reported in other longevity studies conducted in mice. For example, recent studies in C57BL/6 mice show control median lifespan of more than 850 d (28 months) [51], while longevity studies in the 4-way genetically heterogeneous mice show median lifespan of almost 900 d (30 months) [47]. Although there is a longevity study in which Crj:CD-1 mice show a control median survival of less than 18 months [39], further research is warranted to confirm this finding in at least 1 other strain of mice.

In the 1970s, it was reported that increases in PAs were related to neoplastic growth [52]. Since then, many researchers have regarded PAs as carcinogens. However, most of these historical studies were performed to test the effects of PAs on existing tumors or on the growth of tumors after the initiation of neoplastic growth [53], [54]. We have found no evidence that increased intake of PAs promotes oncogenic transformation in normal cells and animals. Furthermore, there are many reports that PAs are indispensable for normal functioning in a diverse group of organisms ranging from cells and bacteria to plants and mammals [55], [56]. We hope that the results presented here encourage more researchers to investigate the diverse and important bioactivities of PA.