The gut microbiota plays an essential role for maintaining human health. This microbial ecosystem regulates many functions of the host, enhancing the production of useful substances such as essential vitamins, aminoacids, and short-chain fatty acids that exert anti-inflammatory and immunomodulatory effects.1–3
Probiotic bacteria are shown to promote endogenous host defense mechanisms. Besides the stimulation of nonimmunological gut defences, such as rebalance of the gut microbiota, probiotics are able to enhance immune responses and promote the intestinal immunologic barrier.4–6
Nowadays a lot of information is available about the healthy properties of probiotics, which are not always based on proven evidence. Moreover, little is known about the precise mechanisms of action underlying these beneficial effects.7–9 cytokines and the T-helper (Th) balance.10–13 14–16 17,18 19 20–22 23–25
Bifidobacteria are gram positive, obligate anaerobic bacteria that inhabit mainly the colon of healthy infants and adults. Soon after birth, up to 90% of all bacteria in fecal samples detected by fluorescence in situ hybridization are bifidobacteria, and they still make up 3% to 5% of the adult microbiota.26,27 28 29–31 32–37
The relative stability of the gut microbiota composition is decreased after a defined threshold age: increasing age, physiological modifications induced by the aging process, and alterations in diet and life style affect the balance of the intestinal microbiota significantly.38
Several studies have referred to the features of the aged gut microbiota.39,40 41
A recent study by Drago et al42 Bifidobacterium longum in the gut seemed to be a peculiar feature of Centenarians. In particular, 14 strains of B. longum were isolated, characterized, and deposited at an International Collection in the view of further investigations.
The purpose of the present study is to analyze the influence of 5 B. longum strains (DLBL) isolated from healthy Centenarians on the human immune system, particularly through the modulation of cytokine release.
MATERIALS AND METHODS
Bacteria and Growth Conditions
Table 1 reports the list of B. longum isolated from fecal samples of healthy Centenarians by Drago et al.42
TABLE 1: List of the Bifidobacterium longum Probiotic Strains Isolated From Centenarians
Five of these strains, by Probiotical SpA collection, have been used in the present study: B. longum DLBL07 (DSM 25669), B. longum DLBL08 (DSM 25670), B. longum DLBL09 (DSM 25671), B. longum DLBL10 (DSM 25672), and B. longum DLBL11 (DSM 25673). Probiotic strains were stored in 20% glycerol at −80°C. More than 90% of the cells were alive upon thawing. Before use, B. longum was grown in anaerobic jars with CO2 -generating kits (Anaerocult A; Merck, Darmstadt, Germany) overnight at 37°C in de Man-Rogosa-Sharpe broth containing 0.05% cysteine hydrochloride, and then subcultured until the mid-log phase. For the enumeration of live bacteria, the BD Cell Viability Kit with BD Liquid Counting Beads (BD Biosciences, Milano, Italy) were used as instructed by the manufacturer. For stimulation experiments, bacteria were suspended in RPMI-1640 medium [Invitrogen, San Giuliano M.se (MI), Italy] and added to human peripheral blood mononuclear cell (PBMCs) cultures.
Isolation and Stimulation of Human PBMCs
PBMCs were isolated by centrifugation over a Ficoll-Hypaque (Lymphoprep) density gradient from freshly collected, leukocyte-rich buffy coats provided by the local Blood Transfusion Service (Novara, Italy). Cells were washed and adjusted to 1×106 cells/mL in RPMI-1640 supplemented with 2 mM L-glutamine, 5 mM Hepes, and 10% heat-inactivated fetal calf serum. PBMCs were plated in 24-well flat-bottomed polystyrene microtiter plates in the absence or the presence of different stimulants at 37°C and 5% CO2 for 24 and 96 hours. Nonstimulated PBMCs were assessed as controls of basal cytokine production. PBMCs were stimulated with live bacteria in a 1:1 ratio, used either as individual strains or as a mixture.
Purified lipopolysaccharide from Escherichia coli 055:B5 (Sigma Chemicals, Milano, Italy) was used to stimulate PBMCs at a concentration of 10 μg/mL as a 24-hour positive control. The polyclonal T-cell stimulator phytohemagglutinin (PHA-P; Sigma Chemicals) was used to stimulate PBMCs at a concentration of 10 μg/mL as a 5-day positive control. Cultures were performed in triplicate, and each experiment was performed with PBMCs from 8 donors.
Cytokine Assays
To find out whether the B. longum strains would differ in their ability to induce the cytokine pattern, human PBMCs were incubated with 5 different B. longum DLBL strains used together (DLBL mix) or alone.
The cytokine concentration of the supernatants was measured using specific human enzyme-linked immunosorbent assay Ready-SET-Go! Kit (eBioscience, San Diego, CA), according to the manufacturer’s instructions. Levels of IL-12p70 were measured in the cell culture supernatants after 24 hours of incubation. Levels of IFN-γ, IL-4, and IL-17A were detected after 5 days. The sensitivity of assays for each cytokine was as follows: 2 pg/mL for IL-4 and 4 pg/mL for IFN-γ, IL-12p70, and IL-17A.
Statistical Analysis
Results are expressed as the means±SE of the mean of duplicates measures determined in 8 independent experiments. Significant differences between unstimulated and stimulated samples were tested using the t test with GraphPad Prism 6.0 software. Values of P <0.05 were considered significant.
RESULTS
Effects of Live Bacteria on Human Cytokine Production
As shown in Table 2 , individual B. longum DLBL strains and their association effectively induced cytokine synthesis. A comparison with the cytokine profiles induced by other B. longum strains not isolated from Centenarians as well as data from other probiotic species and genera are reported.
TABLE 2: Cytokine Profiles Induced by the Bifidobacterium longum Strains Isolated From Centenarians Compared With Additional B. longum Strains Isolated From Adults and With Other Probiotic Genera and Species
All DLBL bacteria strains have the capacity to modulate significantly IFN-γ production, but the magnitude of Th1-type cytokine production varied depending on the DLBL strain (range, 170 to 290 pg/mL). The level of IFN-γ induced by B. longum DLBL was lower than that of the positive control. All B. longum DLBL strains had no effect on the production of IL-12p70. Analysis of IL-4 modulation shows that all strains modulate Th2 cytokine release significantly (range, 25 to 30 pg/mL) (Fig. 1 ).
FIGURE 1: Cytokine production after 4 days in human peripheral blood mononuclear cells stimulated by individual Bifidobacterium longum DLBL or their mixture. *P <0.05, **P <0.01, ***P <0.001. IL indicates interleukin; IFN, interferon; PHA, phytohemagglutinin.
The modulation of cytokine secretion induced by the DLBL mixture was similar to the results obtained with individual strains, except for IL-17A.
PBMC incubation with individual B. longum DLBL07, B. longum DLBL08, B. longum DLBL09, B. longum DLBL10, and B. longum DLBL11 demonstrated a significant increase in the release of IFN-γ by 15, 9, 12, 11, and 12 times, respectively, compared with unstimulated PBMCs (Table 2 ). PBMC treatment with the DLBL mixture showed a further significant increase in the release of Th1-type cytokine by 16 times, compared with the unstimulated control (Table 2 ). Both the individual B. longum DLBL strains and their mixture induced a significant increase in the release of IL-4 by about 3 times (range, 3.1 to 3.7), compared with unstimulated PBMCs.
DISCUSSION
There are many well-known studies concerning the effect of ethnicity, genetic heritage, food habits, and environment on the health of Centenarians.39,43,44 42
In our study, one of the most remarkable aspects is that all the strains used have some biological similarities, as they are able to stimulate equally IFN-γ, IL-4, IL-12p70, and IL-17A, both alone and in association (Table 2 and Fig. 1 ). This evidence may be related to the fact that these B. longum from Centenarians have some similar biological peculiarities.
A further confirmation is that B. longum strains isolated from different populations have cytokines induction profiles different from each other and from those typical of strains collected from Centenarians (Table 2 ).
The fact that all samples were obtained from Centenarians itself demonstrates a perfect homeostasis. Microbiota may be a part of this process, together with ethnicity, genetic heritage, food habits, and environment, and the similar biological behavior of their B. longum may confirm this argumentation.
It is now very important to continue to study the microbiota of Centenarians with the aim to find other biological links.
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