Oral supplementation with Lactobacillus delbrueckii subsp. bulgaricus 8481 enhances systemic immunity in elderly subjects

Abstract

Throughout life, there is an aging of the immune system that causes impairment of its defense capability. Prevention or delay of this deterioration is considered crucial to maintain general health and increase longevity. We evaluated whether dietary supplementation with Lactobacillus delbrueckii subsp. bulgaricus 8481 could enhance the immune response in the elderly. This multi-center, double-blind, and placebo controlled study enrolled 61 elderly volunteers who were randomly assigned to receive either placebo or probiotics. Each capsule of probiotics contained at least 3 × 10(7) L. delbrueckii subsp. bulgaricus 8481. Individuals in the study were administered three capsules per day for 6 months. Blood samples were obtained at baseline (time 0), end of month 3, and month 6. We characterized cell subpopulations, measured cytokines by flow cytometry, quantified T cell receptor excision circle (TREC) by real-time PCR (RT-PCR), and determined human β-defensin-2 (hBD-2) concentrations and human cytomegalovirus (CMV) titers by enzyme-linked immunosorbent assay (ELISA). Elderly responded to the intake of probiotic with an increase in the percentage of NK cells, an improvement in the parameters defining the immune risk profile (IRP), and an increase in the T cell subsets that are less differentiated. The probiotic group also showed decreased concentrations of the pro-inflammatory cytokine IL-8 but increased antimicrobial peptide hBD-2. These effects disappeared within 6 months of stopping the probiotic intake. Immunomodulation induced by L. delbrueckii subsp. bulgaricus 8481 could favor the maintenance of an adequate immune response, mainly by slowing the aging of the T cell subpopulations and increasing the number of immature T cells which are potential responders to new antigens.

Fig. 1

Flowchart of participants through trial

Fig. 2

CD4/CD8 ratio, percentage of CD8+CD28^null, and CMV antibody titer in elderly from the placebo and probiotic groups. a CD4/CD8 ratios were analyzed and compared between the two groups at 0, 3, and 6 months post-initiation of probiotic or placebo consumption. Staining was performed with anti-CD3-FITC, anti-CD4-APC, and anti-CD8-PerCP to gate CD4+ and CD8+ T cells. CD4/CD8 ratio less than 1.0 was used to identify individuals with an IRP. b Percentages of CD8+ T cells lacking CD28 expression in peripheral blood of elderly. Whole blood was stained with anti-CD3-FITC, anti-CD28-PE, and anti-CD8-PerCP. Frequencies of CD28^null cells in gated CD3+CD8+ lymphocytes were quantified. c Serum anti-CMV antibody titer was measured by ELISA at baseline and 6 months and was compared. Patient samples are quantified and interpreted by comparing the cut-off index ratio (Cutoff Index = OD value of sample/Cut-off value). A ratio of 1.0 is equivalent to the cut-off value. Cutoff index > 1.1 was considered positive, and the result of this ratio is a semi-quantitative titer. Outlier and extreme values, which were calculated by adding 1.5 and 3 times the IR to the 75th percentile, were represented by circles and stars, respectively. Paired t-test was used to compare values between groups, and p-values are depicted in the panels

Fig. 3

Distribution of T cell subsets in the probiotics group. CD4+ and CD8+ T cells were categorized into NAÏVE, CM, EM, and EMRA cells. Distribution of EMRA in CD4+ and CD8+ T cells into subsets defined by CD28 and CD27 expression. Expression of CD45RA, CCR7, CD27, and CD28 was analyzed by flow cytometry in isolated CD4+ and CD8+ T cells from the two groups of elders at 0, 3, and 6 months. Histograms represent percentage of a CD4+ and b CD8+ T cells in each subset (NAÏVE, CM, EM, and EMRA) in the groups of elderly (time 0: white bars, time 3 months: gray bars, time 6 months: black bars). c, d Individual segments of the pie charts represent the proportions of cells with each combination of CD28 and CD27 in the EMRA CD4+ (c) and CD8+ (d) T cell subsets. EMRA can be divided into pE1 (CD27 + CD28+) and pE2 (CD27 + CD28^null, only in CD8 T cells) and E (CD27^nullCD28^null). Significant differences between subsets related to total CD4+ and CD8+ T cells are indicated (Paired t-test). Each bar in the histograms represented the mean ± SEM

Fig. 4

Proximity to the thymus of the T cell subsets and TREC content. Whole blood from the elderly of the probiotic group was stained with anti-CD45RA-FITC, anti-CD31-PE, and these CD4+ and CD8+ T cell subsets were evaluated by flow cytometry. a Representative dot-plots showing the frequency of CD45RA+CD31+ in CD4+ and CD8+ T cells from elderly. Percentage of positive cells in each subpopulation in this representative experiment is expressed in the right side, and summarized results from all donors (mean and SD) were also expressed in dot-plots. b The TREC content was measured in PBLs cells from elders belonging to placebo/probiotic group. TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate, and bars represented results from the grouped elders (mean ± SEM)

Fig. 5

Quantification of pro-inflammatory cytokine IL-8 and hBD-2 antimicrobial peptide in elderly in the placebo/probiotic groups at 0, 3, and 6 months. a Serum IL-8 concentrations into the placebo and probiotic groups were measured by flow cytometry. b Quantification of the hBD-2 peptide in serum from the elderly in the two groups by ELISA. Outlier values were represented by circles and extreme values by stars, calculated by adding 1.5 and 3 times the IR to the 75th percentile, respectively. The Wilcoxon non-parametric method was used to compare frequencies in the groups. P-values are depicted in the panels

Fig. 6

Changes in percentage in NK cells, TREC content, T cell subsets distribution, and percentage of CD45RA+CD31+ into CD4+ and CD8+ T cells 6 months after stopping the probiotic intake. Measurements were made in nine elderly people at baseline, 6 months from the beginning of the study, and 6 months after stopping the probiotic intake. a Percentages of CD16+56+ cells with respect to the total CD45+ cells were compared throughout the study. Staining was performed with “Multiset CD3-FITC/CD16+56-PE/CD45-PerCP/CD19-APC,” and frequencies of CD16+56 cells in gated CD45+ subsets were analyzed. b TREC content in elderly T cells. The TREC content was measured in T cells from elders, and TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate. c, d Distribution of CD4+ and CD8+ T cells into NAÏVE, CM, EM, and EMRA. Expression of CD45RA and CCR7 was analyzed by flow cytometry in isolated CD4+ and CD8+ T cells. e, f Frequency of CD45RA+CD31+ in CD4+ and CD8+ T cells from elderly. Whole blood from the elderly of the probiotic group was stained with anti-CD45RA-FITC, anti-CD31-PE, and anti-CCR7-APC; and CD4+ and CD8+ T cells were evaluated by flow cytometry. Bars in the histograms represented the mean ± SEM. The Paired t-test (when data were normally distributed) and Wilcoxon non-parametric method (when data were not normally distributed) were used to compare frequencies between groups. *Significant differences with values obtained 6 months after beginning the probiotic intake (p < 0.05)