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Keywords:

  • Lactobacillus gasseri;
  • immunosenescence;
  • natural killer cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present study was conducted to test the ability of probiotic lactobacilli to alter age-related immunosenescence in host animals. Senescence-accelerated mouse prone 1 mice were orally fed heat-killed Lactobacillus gasseriTMC0356 (TMC0356) for 4 and 8 weeks at dosages of 10 mg day−1 after a 16-week period of prefeeding with a standard diet. After 4 and 8 weeks of TMC0356 intervention, splenic activation of natural killer (NK) cells and mRNA expression of cytokines and other immune molecules in the lungs were analysed. After 4 and 8 weeks, splenic NK cell activities were significantly higher in the TMC0356-fed mice compared with control mice (P < 0.05). After 4 weeks, mRNA expression of interleukin-2 and interferon-(α and β) receptor 1 in lung cells isolated from the TMC0356-fed mice also increased significantly compared with that in lung cells from the control mice (P < 0.05). These results suggest that lactobacilli, especially certain selected strains, might enhance cell-mediated immunity in host animals and thereby alter age-related immunosenescence.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Immunosenescence is defined as the state of deregulated immune function that contributes to the increased susceptibility of the elderly to infection, and possibly to autoimmune diseases and cancer (Ginaldi et al., 1999). When immunosenescence occurs, the functional capacity of the immune system of the host gradually declines with age. The most dramatic changes in the immune system that occur with age involve the T-cell compartment, the arm of the immune system that protects against pathogens and tumors (Ginaldi et al., 1999; Castle, 2000). The fact that T lymphocytes are more severely affected than B cells or antigen-presenting cells is primarily a result of involution of the thymus, which is almost complete at the age of 60 years. The host then becomes dependent on T cells of various specificities, which eventually leads to changes in the T-cell repertoire. CD45RA+ ‘native’ cells are replaced by CD45RA ‘memory’ cells and T-cell receptor oligoclonality develops. Simultaneously, T cells with signal transduction defects accumulate. Age-related T-cell alterations lead to a decreased clonal expansion and a reduced efficiency of T-cell effector functions such as cytotoxicity or B-cell help. Decreased antibody production and a shortened immunological memory are the consequence and severity of disease. Efficient protection of elderly individuals by suitable vaccination strategies is therefore a matter of great importance (Grubeck-Loebenstein, 1997; Effros, 2001).

Interleukin (IL)-12 is a cytokine produced by mononuclear phagocytes and dendritic cells that serve as mediators of the innate immune response to intracellular microbes; it is a key inducer of cell-mediated immune responses to microbes (Peakman & Vergani, 1997). IL-12 activates natural killer (NK) cells, promotes interferon (IFN)-γ production by NK and T cells, enhances the cytolytic activity of NK cells and cytolytic T lymphocytes, and promotes Th1 cell development. Many studies have indicated that Gram-positive bacteria, especially lactobacilli, and their cell-wall compounds are potent inducers of IL-12 for human monocytes (Haller et al., 2000; Hessle et al., 2000; Gill et al., 2001).

In the present study, heat-killed Lactobacillus gasseri TMC0356 (TMC0356) cells were tested to determine their ability to alter age-related immunosenescence using short-lived senescence-accelerated mouse prone 1 (SAMP1) as a test model.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

TMC0356 was stored at the Technical Research Laboratory of Takanashi Milk Products Co., Ltd (Yokohama, Japan). Lactobacilli were routinely cultured at 37 °C for 18 h in modified MRS (deMan, Ragosa and Sharpe) broth. After incubation, the bacteria were collected by centrifugation (7000 g) and washed with sterile 0.85% NaCl. After washing, the collected bacteria were killed by heat treatment at 90 °C for 5 min in sterile 0.85% NaCl. The heat-killed bacteria were lyophilized and kept at −80 °C until use. The viable count of lyophilized bacteria was < 100 CFU g−1 on MRS agar plates (below detection limits). Total counts in the heat-killed bacteria were more than 1.0 × 1011 CFU g−1, calculated using microscopy.

Mouse experiment

A schematic of the mouse experiment is shown in Fig. 1. For the experiment, 15-week-old male SAMP1 mice were purchased from Japan SLC (Hamamatsu, Japan). The mice were housed in plastic cages under a 12-h light–dark cycle, allowed free access to tap water ad libitum, fed a standard diet (CRF-1; Oriental Yeast Co., Tokyo, Japan) for 7 days and randomly divided into two groups (control and TMC0356 fed/test) of 36 mice each. Thirty-six test mice were orally administered 10 mg of lyophilized TMC0356 in 200 μL of sterile physiological saline each day for 4 weeks (18 test mice) or 8 weeks (18 test mice). In addition, 36 control mice were orally administered 200 μL of sterile physiological saline each day for 4 weeks (18 mice) or 8 weeks (18 mice). All experiments were performed in accordance with the guidelines for laboratory animal care of Oriental Yeast Co. and Takanashi Milk Products, Co., Ltd.

image

Figure 1. Experimental schematic.

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NK cell activity

After 4 and 8 weeks of oral administration of TMC0356, the test mice were sacrificed and their spleens were removed aseptically. Isolated spleen cells were analysed for NK cell cytotoxicity (NK cell activity), as described by Hosokawa et al. (1987ab) with some modifications. Briefly, NK cell activity was determined by a 51Cr release assay using 51Cr-labeled YAC-1 cells as target. A total of 5 × 106 spleen cells were mixed with 1 × 105 target cells in 96-well microculture plates at an effector-to-target ratio of 50 : 1 in a total volume of 0.2 mL of RPMI 1640 medium containing 10% fetal bovine serum. The plates were incubated at 37 °C in 5% CO2. After 4 h of incubation, 100 μL of supernatant from each well was harvested by centrifugation (680 g, 4 min), and radioactivity in the supernatant was determined using an ARC-370M gamma counter (Aloka Co., Ltd., Tokyo, Japan). Cytotoxicity as a percentage of specific 51Cr release was calculated as follows: Cytotoxicity (%) = (ER − SR)/(MR − SR) × 100, where ER is experimental release, SR is spontaneous release and MR is maximum release.

Real-time quantitative RT-PCR of mRNA expression

To obtain lung specimens, the mice were sacrificed and their lungs were removed aseptically. Large tissue samples of ≤ 0.5 cm in any single dimension were cut from the lungs, immersed in 5–10 volumes of RNAlater solution (Ambion Inc., TX), and stored at 4 °C overnight. After overnight incubation, the samples were stored at −80 °C.

Total RNA was isolated using a FastPure RNA kit (Takara Bio Inc., Otsu, Japan). Reverse transcription was performed using a PrimeScript RT reagent kit (Takara Bio Inc.). IFN-α1, IFN-γ, IFN-(α and β) receptor 1 (IFN-ar1), perforin 1, CD247 antigen, killer cell lectin-like receptor subfamily k, member 1 (Klrk1), tumor necrosis factor (TNF), IL-12a, IL-12 receptor β 1 (IL-12rb1), IL-2, IL-2 receptor β (IL-2rb), IL-5, IL-6, IL-17 and IL-21 mRNA levels were determined by real-time quantitative RT-PCR using SYBR green I (SYBR Premix Ex Taq II; Takara Bio Inc.).

Fluorescent signals were detected using a Thermal Cycler Dice Real-Time System TP800 (Takara Bio Inc.), and primers were designed using the Perfect Real Time Support System (Takara Bio Inc.).

The primers used in the present study were as follows: for IFN-γ (forward) 5′-CGGCACGTCATTGAAAGCCTA-3′, (reverse) 5′-GTTGCTGATGGCCTGATTGTC-3′; for IFN-α1 (forward) 5′AGCCATCCCTGTCCTGAGTG-3′, (reverse) 5′-TCATTGAGCTGCTGGTGGAG-3′; for IFN-ar1 (forward) 5′-CCATGAGTGACACCTTGCTTGTTTA-3′, (reverse) 5′-AGGGTGAACTCTGGGCCATC-3′; for Prf1 (forward) 5′-TTCGGGAACCAAGCTACACCA-3′, (reverse) 5′-CAGGCTGTAGTCCACCAGACCA-3′; for Cd247 (forward) 5′-CTGCTGGATCCCAAACTCTGCTA-3′, (reverse) 5′-GTTGGCAGCAGTCTCTGCACTC-3′; for Klrk1 (forward) 5′-AATTACGACCTCAAGCCAGCAAAG-3′, (reverse) 5′-CAAGGCTATAGCAAGGACTCGAACA-3′; for TNF (forward) 5′-AAGCCTGTAGCCCACGTCGTA-3′, (reverse) 5′-GGCACCACTAGTTGGTTGTCTTTG-3′; for IL-12a, (forward) 5′-TGTCTTAGCCAGTCCCGAAACC-3′, (reverse) 5′-TCTTCATGATCGATGTCTTCAGCAG-3′; for IL-12rb1 (forward) 5′-TGGAGTCTCGGCTTGGGAAAC-3′, (reverse) 5′-CACATTCCAGTCCATTCGCAAC-3′; for IL-2 (forward) 5′-GGAGCAGCTGTTGATGGACCTAC-3′, (reverse) 5′-AATCCAGAACATGCCGCAGAG-3′; for IL-2rb (forward) 5′-TTGCATGTGGAGCCATGAAGA-3′, (reverse) 5′-ACCCGAGGATCAGGTTGCAG-3′; for IL-17a (forward) 5′-ACGCGCAAACATGAGTCCAG-3′, (reverse) 5′-AGGCTCAGCAGCAGCAACAG-3′; for Actb (forward) 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, (reverse) 5′-ATGGAGCCACCGATCCACA-3′. The procedure for real-time quantitative RT-PCR was 30 s at 95 °C, followed by 45 cycles of 5 s at 95 °C and 30 s at 60 °C. Analysis was performed with a Thermal Cycler Dice Real Time System TP800 2.01C (Takara Bio Inc.) and normalized by against actin-β.

Statistical analysis

Statistical comparisons between the three groups were made using the Tukey–Kramer test. Statistical significance of differences between the two groups was calculated using an unpaired Student's t-test or Welch's t-test after performing an F-test. Differences were considered significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Body weight

The body weight of test mice fed with TMC0356 was increased as did those in control group. After 4 and 8 weeks, there were no significant differences in body weight among the experimental groups.

NK activity of mice splenocytes

Cytotoxicities of isolated spleen cells from the test mice are shown in Fig. 2. After 4 weeks of oral administration of TMC0356, NK cell activity was significantly higher in the test mice than in the control mice (6.1 ± 0.5 vs. 4.8 ± 0.3; P < 0.05). After 8 weeks of oral administration of TMC0356, NK cell activity was still significantly higher in the test mice than in the control mice (6.3 ± 0.9 vs. 4.2 ± 0.3; P < 0.05). NK activity of spleen cells isolated from the control mice after 8 weeks decreased compared with that after 4 weeks. However, this difference was not statistically significant (= 0.15).

image

Figure 2. Effect of oral administration of lactobacilli on NK activity of splenocytes. Results are expressed as the mean ± SEM for each group (n = 18).

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mRNA expression of cytokines and immune molecules in the lungs

Pulmonary mRNA expression of cytokines and immune molecules in the lungs of the test mice was also analysed (Fig. 3). After 4 weeks, pulmonary mRNA expression of IL-2 and IFN-ar1 was significantly higher in the test mice than in the control mice (P < 0.01). Pulmonary mRNA expression of IL-12a and IL-12rb1 tended to be higher in the test mice than in the control mice. However, such changes were not statistically significant (= 0.074 and 0.068, respectively).

image

Figure 3. mRNA expression of cytokines and immune molecules in the lungs of mice. Results are expressed as the mean ± SEM for each group (n = 18). The housekeeping Actb gene was used in the same sample to represent the relative mRNA level.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

TMC0356 is a new probiotic strain of L. gasseri that was originally isolated from the intestine of a healthy human adult (Hosoda et al., 1998). This bacterium has expressed strain-dependent immune regulatory effects such as apparent simulation of IL-12 production from macrophages in cell line and animal studies (Morita et al., 2002; Harata et al., 2009; Kawase et al., 2009). In several recent animal and human studies, TMC0356 significantly improved allergic symptoms in patients with Japanese cedar pollinosis and in ovalbumin-immunized animals, protected host animals from influenza virus infection, and significantly suppressed the growth of translated tumors (Kawase et al., 2006, 2007ab, 2009; Harata et al., 2009; Wang et al., 2009). These health-promoting effects of TMC0356 are believed to be partly a result of a strain-dependent regulatory effect on cell-mediated immunity (CMI) of host animals characterized by elevated IFN-γ production and increased Th1-type immunity. Recently, some selected Lactobacillus and Bifidobacterium strains with properties that bolster CMI have been found to possess potent health-promoting effects against various age-associated physiological changes such as the development of osteoporosis (Kimoto-Nira et al., 2007, 2009). In light of these findings, we hypothesized that TMC0356 might positively alter the immunosenescence of aged host animals by stimulating their CMI, and consequently might improve the natural defense of aged host animals against various infections.

SAM is a well-known murine model of accelerated senescence. SAM consists of SAMP (prone) and SAMR (resistant) lines. SAMP lines are characterized by the accumulation of senile features as well as earlier onset and faster progress of age-related pathological phenotypes, such as amyloidosis, impaired immune responses, senile osteoporosis, and deficits in learning and memory (Hanada et al., 1991). Furthermore, age-related early loss of immune function has been clearly demonstrated in SAMP strains such as profound defects in the antibody response to a TD antigen, early onset of regression and a sharp decline in NK cell activity from the level in the control mice at 2 months of age (Hosokawa et al., 1987ab).

In the present study, splenic activation of NK cells of the control SAMP1 mice decreased with age from 20 to 24 weeks (between 4 and 8 weeks of oral administration of saline). These results are in good agreement with the findings of previous studies indicating that NK activities of spleen cells could decline significantly in SAMP1 mice aged 2 months, an age-related change in immune activity (Hosokawa et al., 1987ab). Splenic NK activities in the test SAMP1 mice orally fed TMC0356 were significantly higher than those in the control mice at 4 and 8 weeks. Furthermore, the test SAMP1 mice showed almost the same splenic activities at 19 and 23 weeks. These results indicate that oral administration of TMC0356 can enhance CMI in SAMP1 mice, increase NK activities of spleen cells in vitro and alter the decline in age-related changes in NK activities in this model animal. These results suggest that oral administration of lactobacilli, especially some selected strains such as TMC0356, can improve immunosenescence in elderly humans. To the best of our knowledge, the present study is the first to demonstrate that oral administration of lactobacilli can alter age-related loss of immune function.

In previous studies, orally administered TMC0356 protected mice from H1N1 influenza virus infection (Kawase et al., 2010). In addition, oral administration of heat-killed TMC0356 also significantly protected the mice from influenza virus infection (our unpublished data). Furthermore, these protective effects are considered to result from oral administration of the heat-killed TMC0356, which stimulates respiratory immune responses; these effects are characterized by upgraded mRNA expression of cytokines and other immune molecules in the lungs (our unpublished data). In the present study, orally administered TMC0356 significantly increased mRNA expression of IL-2 and IFN-α and -β in the SAMP1 mice. IL-2 and IFN-α and -β can stimulate the proliferation and differentiation of NK cells (Peakman & Vergani, 1997). Thus, upregulation of mRNA expression of IL-2 and IFN-α in the lungs may contribute to activation of NK cells in the lungs of the TMC0356-fed mice. These results suggest that improvements in immunosenescence resulting from oral administration of TMC0356 can contribute to respiratory immune responses and enhance natural defense against respiratory infections.

SAMP1 mice will develop spontaneously ileal inflammation at the age of about 10–15 weeks (McNamee et al., 2010). The findings of the present study also suggest that oral administration of heat-killed TMC0356 might alter inflammatory bowel disease using SAMP1 as a test model.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank H. Kawasaki and M. Harada of Oriental Yeast Co. for their technical help with animal experiments. This study was supported by a Grant-in-Aid for Research and Development from the Japanese Ministry of Agriculture and Forestry.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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