Figure 5. Protective effect of Lactobacillus paracasei KW3110 on age-induced histological changes and ganglion cell loss in the retina. ( A ) Hematoxylin and eosin staining of retinal sections in young mouse (3-months-old) fed a control (CTL) diet and aged mice (22-months-old) fed a diet either with or without L. paracasei KW3110 (KW3110 diet). Arrow heads indicate the ganglion cell layer (GCL) and outer nuclear layer (ONL), respectively. Scale bar represents 100 μm. ( B ) The survival rate of retinal ganglion cells (RGCs) in aged mice (22-months-old) fed a diet either with or without L. paracasei KW3110 (KW3110 diet) were analyzed compared to the survival of RGCs in young mice (3-months-old) fed a control diet. Values are presented as the means ± SEM. Significance was assumed if the p value was < 0.05. ** p < 0.01. ( C ) ONL thickness was lower in aged mice (22-months-old) fed control diet than in aged mice fed a diet with L. paracasei KW3110. Values are presented as the means ± SEM. Significance was assumed if the p value was < 0.05. * p < 0.05; ** p < 0.01.

To elucidate the effects of the intake of L. paracasei KW3110 on age-related inflammatory phenotypes in retina, the number of RGCs was counted ( Fig. 5A ). The survival of RGCs was significantly decreased in aged mice as compared with that of young control mice. However, the survival of RGCs was significantly improved in aged mice fed a diet containing L. paracasei KW3110 from 16–22 months of age for 6 months ( Fig. 5A, B ), as compared with age-matched mice fed a control diet. In addition, the outer nuclear layer (ONL) thickness, corresponding to the photoreceptor layer, in aged mice fed a diet containing L. paracasei KW3110 for 6 months was also significantly thicker than in age-matched mice fed a control diet. These results were consistent with the suppressive results of proinflammatory cytokine levels in aged mice fed a diet containing L. paracasei KW3110.

Figure 4. Intake of Lactobacillus paracasei KW3110 mitigated retinal inflammation. Intake of L. paracasei KW3110 in aged mice (17-months-old) suppressed the expression of inflammatory cytokines in retinal macrophage of aged mice. F4/80 and CD11b-positive macrophage in retina were gated as shown in ( A ), and median fluorescent intensity of intracellular IL-6, IFN-γ, and TNF-α were analyzed by flow cytometry ( B - D ). Significance was assumed if the p value was < 0.05; * p<0.05. All abbreviations are defined in the Figure 3 legend.

As the lower serum levels of proinflammatory cytokines in aged mice were seemingly related to the suppression of age-related inflammatory phenotypes in peripheral tissues, we investigated whether intake of L. paracasei KW3110 also mitigated age-related retinal inflammation. Intake of L. paracasei KW3110 from 11–17 months of age for 6 months, in aged mice, significantly decreased the expression of IFN-γ and interleukin-6 (IL-6) in CD11b-positive and F4/80-positive retinal immune cells and macrophage ( Fig. 4A-C ) as compared with in age-matched mice fed a control diet. The expression of tumor necrosis factor-α (TNF-α) in CD11b-positive and F4/80-positive retinal macrophage was also lower than in age-matched mice fed a control diet ( Fig. 4D ).

Figure 3. The levels of proinflammatory cytokines in the serum were mitigated in aged mice fed a diet containing Lactobacillus paracasei KW3110 as compared with age-matched control mice. Serum was collected and subjected to multiplex analyses to determine levels of cytokines (IL-1β, IL-6, IL-13, IL-17, IFN-γ, TNF-α, KC, and MCP1) in young (3-months-old) and aged mice (22-months-old). Values are presented as the means ± SEM. Significance was assumed if the p value was < 0.05. * p < 0.05; ** p < 0.01. CTL = control diet; KW3110 = Lactobacillus paracasei KW3110 diet; IL = interleukin; IFN = interferon; TNF = tumor necrosis factor; KC = keratinocyte chemoattractant; MCP1 = monocyte chemoattractant protein 1.

The age-related inflammatory phenotypes in various tissues are associated with the serum levels of proinflammatory cytokines, which are produced from inflammatory immune cells. Therefore, we evaluated serum levels of proinflammatory cytokines and chemokines in aged mice. As shown in Fig. 3 , the serum levels of proinflammatory cytokines and chemokines in aged mice were higher than those in control young mice ( Fig. 3 ). Interestingly, serum levels of some cytokines and chemokines, interleukin-17 (IL-17), keratinocyte chemoattractant (KC), and interleukin-13 (IL-13), were significantly lower in aged mice fed a diet containing L. paracasei KW3110 from 16 months of age to 22 months of age for 6 months, than in age-matched mice fed a control diet ( Fig. 3 ). The concentrations of the other proinflammatory cytokines were also lower in aged mice fed a diet containing L. paracasei KW3110 for 6 months. These changes of proinflammatory cytokine levels could be observed in aged mice fed a diet containing L. paracasei KW3110 only for 2 months ( Supplementary Fig. 2 ).

Figure 2. Intake of Lactobacillus paracasei KW3110 suppressed the inflammatory CD4-positive T cell expansion in the lamina propia of the small intestine (SI-LP). ( A and B ) To detect inflammatory cytokine-producing cells, SI-LP cells from young mice (3-months-old) and aged mice (17-months-old) were cultured under stimulation with Leukocyte Activation Cocktail plus BD GolgiPlug, and analyzed by flow cytometry. ( A ) Representative data of CD4-positive cells from aged mice fed a diet with (KW3110) or without (CTL) L. paracasei KW3110. ( B ) The ratio of CD3ε- and CD4-positive to live cells. ( C ) Representative data of CD4- and interferon gamma (IFN-γ)-positive cells from aged mice fed a diet with or without L. paracasei KW3110. ( D ) The ratio of CD4- and IFN-γ-positive cells to CD4-positive cells. ( E ) The expressions of programmed cell death protein 1 (PD-1) in CD3ε- and CD4-positive cells were analyzed by flow cytometry. M.F.I. indicates mean fluorescence intensity.

We have previously shown that orally-provided L. paracasei KW3110 interacted with immune cells in the small intestine [ 43 ]. In addition, intake of L. paracasei KW3110 altered gut bacterial flora composition in aged mice ( Fig. 1 ). Thus, to examine the effects of L. paracasei KW3110 on the immune system in the small intestine with aging, 11-month-old mice were fed a diet with or without L. paracasei KW3110 for 6 months. The ratio of CD3ε- and CD4-double positive T cells to live cells and the ratio of interferon-γ (IFN-γ)-producing CD4-positive T cells to CD4-positive T cells, known as indicators of age-related inflammation in SI-LP cells, in aged mice fed a control diet, was higher than that in control young mice ( Fig. 2B, D ). The expression of programmed cell death protein 1 (PD-1), known as an indicator of immune senescence in CD4-positive T cells, in aged mice fed a control diet, was also higher than that in control young mice ( Fig. 2E ). However, the intake of L. paracasei KW3110 for 6 months in aged mice significantly decreased the ratio of CD3ε- and CD4-double-positive T cells to live cells in SI-LP ( Fig. 2A, B ), the ratio of IFN-γ-producing CD4-positive T cells to CD4-positive T cells ( Fig. 2C, D ), and the expression of PD-1 in CD4-positive T cells ( Figure 2E ). In contrast, the ratio of CD4- and Foxp3-positive cells, known as regulatory T cells, in SI-LP was not changed in aged mice fed a diet either with or without L. paracasei KW3110 ( Supplementary Fig. 1 ).

Figure 1CD. The intake of Lactobacillus paracasei KW3110 in aged mice affected the gut microbial composition. ( C ) Distribution of gut microbiota (% of total 16S rDNA) at the family level. Families with proportions less than 1% are not listed. ( D ) Comparisons of relative abundances of Peptostreptococcaceae (left panel), Bifidobacteriaceae (middle panel), and Streptococcaceae (right panel) families. Values are presented as the means ± SEM. Significance was assumed if the p value was < 0.05. * p < 0.05, ** p < 0.01.CTL = control diet; KW3110 = Lactobacillus paracasei KW3110 diet

At the bacterial family level, the bacterial ratios in the feces were altered in aged mice as compared with young control mice ( Fig. 1C ). In the aged mice groups, the intake of L. paracasei KW3110 affected some bacterial abundances. For example, the mean relative abundances of Peptostreptococcaceae ( p = 0.011) and Bifidobacteriaceae ( p = 0.038) were significantly higher in aged mice fed a diet containing L. paracasei KW3110 for 6 months than in age-matched mice fed a control diet ( Fig. 1D ). In contrast, the mean relative abundance of Streptococcaceae was significantly lower ( p = 0.0079) in aged mice fed a diet containing L. paracasei KW3110 than in age-matched mice fed a control diet ( Fig. 1D ).

Figure 1AB. The intake of Lactobacillus paracasei KW3110 in aged mice affected the gut microbial composition. Feces were collected and subjected to flora analysis in young (3-months-old) and aged mice (22-months-old). ( A ) Distribution of gut microbiota (% of total 16S rDNA) at the phylum level. ( B ) Comparison of the Firmicutes to Bacteroidetes ratio. Values are presented as the means ± SEM of relative abundance of each phylum.

The gut microbiota plays a critical role in the immune system, and aging has been reported to alter gut bacterial flora composition [ 35 ]. Previous studies have reported that some prebiotics and probiotics can alter gut bacterial flora composition and improve immune defects [ 44 , 45 ]. Therefore, to investigate whether intake of L. paracasei KW3110 affected the gut microbiota composition in aged mice, 16-month-old mice were fed a diet with or without L. paracasei KW3110 for 6 months. We analyzed bacterial 16S ribosomal RNA gene sequences in the feces. The microbiota composition at the phylum level revealed that the Firmicutes / Bacteroidetes ratio was lower in aged mice fed a control diet than in young mice fed a control diet ( Fig. 1A, B ). This result is consistent with a previous report [ 46 ]. However, in aged mice fed a diet containing L. paracasei KW3110 for 6 months, the Firmicutes / Bacteroidetes ratio was decreased compared with that in age-matched control mice ( Fig. 1B ).

Lactic acid bacteria are widely consumed as probiotics and paraprobiotics to enhance gut barrier function and improve immune systems. Studies have also demonstrated functional roles of several lactic acid bacterial strains in humans, including for the prevention of diarrhea, allergies, and metabolic disorders [ 39 ]. However, the long-term effects of lactic acid bacteria on age-related chronic inflammation remain unclear. We previously reported that Lactobacillus paracasei KW3110 activated macrophages and suppressed excessive inflammation in mice and humans [ 40 – 43 ]. In this study, we demonstrated the suppressive effects of the long-term intake of L. paracasei KW3110 on age-related alterations of gut microbiota composition and expansion of inflammatory CD4-positive T cells in the lamina propria of the small intestine (SI-LP). Furthermore, we also revealed the protective effects of the long-term intake of L. paracasei KW3110 on age-related retinal cell loss. We proposed that the long-term intake of L. paracasei KW3110 contributed to the prevention of chronic inflammation and age-related retinal cell loss in physiologically aged mice.

Age-related immune dysfunctions leading to chronic inflammation have been previously reported. Thymic involution and disruption of homeostatic T cell proliferation, including decreased numbers of naïve T cells, accumulation of memory T cells, and increased numbers of regulatory T cells (Tregs), have been studied [ 22 – 24 ], and altered numbers of B cells with aging, reduced antibody production, and age-related dysfunction of other innate immune cells have also been reported [ 25 – 30 ]. Although some food materials or constituents, for example, prebiotics and probiotics, can improve age-related immune defects [ 31 – 34 ], their mechanism remains poorly understood. Recent studies suggested that the gut microbiota composition may be associated with age-related immune dysfunctions [ 35 – 37 ]. Disruption of gut microbiota composition has been also implicated in retinal diseases, including AMD, through a gut-retina axis [ 38 ]. Therefore, preventive dietary approaches involving alterations of gut microbiota composition for improving age-related retinal chronic inflammation should be studied.

The retina, one of the neural tissues, is also affected by chronic low grade inflammation. Age-related retinal neurodegenerative diseases, such as age-related macular degeneration (AMD), are major causes of blindness in the elderly [ 9 – 12 ]. The disease is caused by age-related retinal cell loss, including retinal ganglion cell (RGC) death [ 13 ] and photoreceptor cell death [ 14 , 15 ], at least partly due to chronic inflammation [ 16 – 19 ]. Several therapeutic pharmacological agents for suppression of retinal diseases have been reported [ 20 , 21 ]. However, human eyes are exposed to daily chronic stress, such as photo-oxidative stress, and as a result, safe and long-term approaches based on diet to mitigate retinal chronic inflammation are especially attractive.

Aging involves a progressive decline of physiological functions in various organs, influenced by several factors, including genetic factors and environmental factors [ 1 – 3 ]. As the aged population has been growing rapidly around the world, the therapeutic and preventive approaches to decelerate senescence are of great concern. Among the features of aging, the decline in immune function has been widely examined, because it results in chronic low grade inflammation, which is a major risk factor for the incidence and prevalence of age-related diseases, including infectious diseases, tumors, and neurodegenerative diseases [ 4 – 8 ].

Discussion

Defective immune functions with aging are key triggers of age-related chronic inflammatory diseases, including infectious diseases, tumors, diabetes, and neurodegenerative diseases [8]. With rapid increases in the aging population, the prevention of age-related immunological dysfunctions and chronic inflammation are necessary to extend the healthy lifespan. In the present study, we demonstrated that long-term intake of heat-killed L. paracasei KW3110 in aged mice significantly enhanced the population of beneficial gut bacteria, of the Bifidobacterium family, and slowed the age-related immune dysfunctions, expansion of the inflammatory IFN-γ-producing CD4-positive T cells in SI-LP, and lowered the serum levels of proinflammatory cytokines. We also found that intake of L. paracasei KW3110 mitigated retinal inflammation and age-related retinal cell death, probably associated with the prevention of age-related retinal diseases.

Age-related alterations of gut microbiota composition have been reported to cause immune senescence and intestinal chronic inflammation [34,47]. Although some probiotics improve the intestinal environment and suppress inflammation, the effects of long-term ingestion of probiotics remain unclear. In the present study, we showed that intake of L. paracasei KW3110 improved the age-related changes of the gut microbiota composition (Fig. 1). The Firmicutes to Bacteroidetes bacterial ratio increased in aged mice fed a control diet as compared with aged mice fed a diet containing L. paracasei KW3110 for 6 months. The age-related increase of Firmicutes to Bacteroidetes ratio was consistent with a previous study [48]. An increased Firmicutes to Bacteroidetes ratio has been reported to be associated with intestinal inflammation in obese patients [49]. The long-term intake of L. paracasei KW3110 might mitigate intestinal inflammation and energy metabolic disorders by modulating the Firmicutes to Bacteroidetes ratio. The intake of L. paracasei KW3110 also increased the relative abundance of Bifidobacteriaceae families (Fig. 1D). Bifidobacterium is known as one of the most beneficial bacterial family, though the bacteria is not detected in the elderly [44,50,51]. In addition, the intake of Bifidobacterium has been reported to result in decreased levels of proinflammatory cytokines, such as TNF-α, in the elderly [44]. In contrast, intake of L. paracasei KW3110 decreased the relative abundance of Streptococcaceae (Fig. 1D). In a previous report, the Streptococcaceae bacteria stimulated the intestinal cells to induce CCL20 chemokine production [52] and inflammatory IFN-γ-producing CD4-positive T cells were attracted by the CCL20 chemokine [53]. In this study, the intake of L. paracasei KW3110 for 6 months in aged mice also significantly reduced Ccl20 gene expression in SI-LP as compared with that of age-matched control mice (Supplementary Fig. 3). These results suggested that the mitigation of age-related alterations in gut microbiota composition by the intake of L. paracasei KW3110 was important to suppress age-related intestinal chronic inflammation.

Indeed, the intake of L. paracasei KW3110 in aged mice significantly suppressed the age-related increase of inflammatory CD4-positive T cells, producing inflammatory cytokines (IFN-γ) at high levels in the SI-LP (Fig. 2). The expression of PD-1, one of the senescence markers, in CD4-positive T cells in SI-LP was also lower in aged mice fed a diet containing L. paracasei KW3110 than that of aged mice fed a control diet. These anti-inflammatory effects on intestinal immune cells might be due to mitigation of age-related decreases of Bifidobacterium. The modulatory effects on intestinal immune cell subpopulations might be associated with a direct interaction between L. paracasei KW3110 and intestinal immune cells. In a previous study, our group showed that L. paracasei KW3110 interacted with intestinal macrophages and suppressed excessive inflammation, including dermatitis, in mice and humans [40–42]. Because lactic acid bacteria, including L. paracasei KW3110, have some toll-like receptor (TLR) ligands, such as lipoteichoic acid, L. paracasei KW3110 might modulate intestinal immune cell activation by a TLR-dependent pathway. Further studies are required to determine the mechanism underlying the relationship between the intake of L. paracasei KW3110 and suppressive effects on age-related expansions of intestinal inflammatory immune cells.

Proinflammatory cytokines produced by intestinal inflammatory immune cells are possibly transferred to other tissues, including the retina, through the blood. Age-related visual function declines and eye diseases, such as AMD, might be associated with retinal inflammation, because retinal chronic inflammation is toxic to retinal cells, including photoreceptor cells and RGCs [16–19]. In the present study, the intake of L. paracasei KW3110 mitigated the inflammation in the retinal macrophage in aged mice (Fig. 4). In addition, the anti-inflammatory effects of L. paracasei KW3110 resulted in inhibition of age-related retinal cell death (Fig. 5). In previous studies, RGCs have been reported to mediate behaviors associated with response to light information and genetic ablation of RGCs results in loss of light-evoked behaviors [54–57]. We have obtained preliminary data that intake of L. paracasei KW3110 in aged mice could preserve the light-evoked locomotor activities as compared with age-matched control mice (data not shown). Although further studies, including immunohistochemical analysis, are needed, L. paracasei KW3110 might suppress age-related retinal cell death. Immunological phenomena are mainly regulated by macrophage in the retina. Macrophage consists of at least two subgroups, classic inflammatory M1 macrophage or alternative anti-inflammatory M2 macrophage [58,59]. M1 macrophages produce inflammatory cytokines, such as TNFα and IL-6, whereas M2 macrophages are considered to be associated with anti-inflammatory responses, including tissue remodeling, through the production of neurotrophic factors and anti-inflammatory cytokines [60,61]. Recently, we found that the intake of L. paracasei KW3110 suppressed light-induced retinal inflammation (unpublished data). Although further studies are required to evaluate the effects of L. paracasei KW3110 on macrophage activation in the retina, the inhibitory effects of L. paracasei KW3110 on age-related retinal cell death might be accompanied at least in part, by the regulation of macrophage activities. In addition, blood-borne macrophages have been reported to enter the retina via the optic nerve and ciliary body in light exposure mice model [62]. Our flow cytometry analysis showed that CD11b and f4/80-positive retinal cells, the retinal macrophages, in aged mice increased more than in young mice (Supplementary Fig. 4). Taken together, proinflammatory macrophages might penetrate into the retina under the age-related retinal degenerative condition.

In the present study, the anti-inflammatory effects of L. paracasei KW3110 on immune cells were observed in aged mice fed each diet from 11–17 months of age for 6 months (Figs. 2 and 4). However, in aged mice fed a diet containing L. paracasei KW3110 from 16–22 months of age for 6 months, such anti-inflammatory effects on immune cells were mild and not significantly different as compared with that of age-match control mice (data not shown). These results suggested that 22 months of age was too old to evaluate the effects of L. paracasei KW3110 on intestinal immune cells.

Lactic acid bacteria are considered to be phagocytosed by intestinal M cells. In a previous report, M cells in aged mice (18 months of age) were not fully functional [63]. This may be because the effects of L. paracasei KW3110 on immune cells in aged mice of 22 months of age were milder than in aged mice of 17 months of age. In the present study, we showed that intake of L. paracasei KW3110 from 16–22 months of age for 6 months significantly suppressed the serum levels of proinflammatory cytokines, alteration of gut microbiota composition, and retinal cell loss (Figs. 1, 3, and 5). Because the anti-inflammatory effects on serum cytokine levels were observed in aged mice fed a diet containing L. paracasei KW3110 (Supplementary Fig. 2), it was suggested that these phenotypes were reflected by the accumulation of anti-inflammatory effects for several months. In other words, continuous preventive methods, like dietary supplementation, might be much more effective in the delay of chronic inflammation.

In conclusion, the intake of L. paracasei KW3110 mitigated chronic inflammation in the intestine and retina, and reduced age-related retinal cell death. Further studies are needed to evaluate the effects in age-related senescent changes of the retina.